IL289396B2 - Using truncated guide rnas (tru-grnas) to increase specificity for rna-guided genome editing - Google Patents

Description

PCT/US2014/029068 WO 2014/144592 Using Truncated Guide RNAs (tru-gRNAs) to Increase Specificity for RNA-Guided Genome Editing CLAIM OF PRIORITY This application claims the benefit of U.S. Patent Application Serial Nos. 61/799,647, filed on March 15, 2013; 61/838,178, filed on June 21, 2013; 61/838,148, filed on June 21, 2013, and 61/921,007, filed on December 26, 2013. The entire contents of the foregoing are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant Nos. DPI GM105378 awarded by the National Institutes of Health. The Government has certain rights in the invention.

TECHNICAL FIELD Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CR1SPR/Cas9 systems, using truncated guide RNAs (tru-gRNAs).

BACKGROUND Recent work has demonstrated that clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems (Wiedenheft et al., Nature 482, 331-338 (2012); Horvath et al., Science 327, 167-170 (2010); Terns et al, Curr Opin Microbiol 14, 321-327 (2011)) can serve as the basis for performing genome editing in bacteria, yeast and human cells, as well as in vivo in whole organisms such as fruit flies, zebrafish and mice (Wang et al., Cell 153, 910-9(2013); Shen et al., Cell Res (2013); Dicarlo et al., Nucleic Acids Res (2013); Jiang et al., NatBiotechnol 31, 233-239 (2013); Jineket al., Elife 2, 600471 (2013); Hwang et al., Nat Biotechnol 31, 227-229 (2013); Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013c); Cho et al., Nat Biotechnol 31, 230-2(2013); Gratz et al., Genetics 194(4): 1029-35 (2013)). The Cas9 nuclease from 5, pyogenes (hereafter simply Cas9) can be guided via base pair complementarity between the first 20 nucleotides of an engineered guide RNA (gRNA) and the complementary strand of a target genomic DNA sequence of interest that lies next to a protospacer adjacent motif (PAM), e.g., a PAM matching the sequence NGG or NAG PCT/US2014/029068 WO 2014/144592 (Shen et al., Cell Res (2013); Dicarlo et al., Nucleic Acids Res (2013); Jiang ct al., Nat Biotechnol 31, 233-239 (2013); Jinek et al., Elife 2, e00471 (2013); Hwang et al., Nat Biotechnol 31,227-229 (2013); Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013c); Cho et al., Nat Biotechnol 31, 230-232 (2013);Jinek et al., Science 337, 816-821 (2012)). Previous studies performed in. vitro (Jinek et al., Science 337, 816-821 (2012)), in bacteria (Jiang et al., Nat Biotechnol 31,233- 239 (2013)) and in human cells (Cong et al., Science 339, 819-823 (2013)) have shown that Cas9-mediatcd cleavage can, in some cases, be abolished by single mismatches at the gRNA/target site interface, particularly in the last 10-12 nucleotides(nts) located in the 3 ’ end of the 20 nt gRNA complementarity region.

SUMMARY CRISPR-Cas genome editing uses a guide RNA, which includes both a complementarity region (which binds the target DNA by base-pairing) and a Cas9- binding region, to direct a Cas9 nuclease to a target DNA (see Figure 1). Thenuclease can tolerate a number of mismatches (up to five, as shown herein) in the complementarity region and still cleave; it is hard to predict the effects of any given single or combination of mismatches on activity. Taken together, these nucleases can show significant off-target effects but it can be challenging to predict these sites. Described herein are methods for increasing the specificity of genome editing usingthe CRISPR/Cas system, e.g., using Cas9 or Cas9-based fusion proteins. In particular, provided are tiuncated guide RNAs (tru-gRNAs) that include a shortened target complementarity region (i.e., less than 20 nts, e.g., 17-19 or 17-18 nts of target complementarity, e.g., 17, 18 or 19 nts of target complementarity), and methods of using the same. As used herein, "17-18 or 17-19" includes 17, 18, or 19 nucleotides.In one aspect, the invention provides a guide RNA molecule (e.g., a singleguide RNA or a crRNA) having a target complementarity region of 17-18 or 17-nucleotides, e.g., the target complementarity region consists of 17-18 or 17-nucleotides, e.g., the target complementarity region consists of 17-18 or 17-nucleotides of consecutive target complementarity. In some embodiments, the guideRNA includes a complementarity region consisting of 17-18 or 17-19 nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence. In some embodiments, the target complementarity region consists of 17-18 nucleotides (of target complementarity). In PCT/US2014/029068 WO 2014/144592 some embodiments, the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a selected target sequence. In some embodiments, the complementarity region is complementary to 18 consecutive nucleotides of the complementary strand of a selected target sequence.In another aspect, the invention provides a ribonucleic acid consisting of the sequence:(X17-18 0rX!7-19)GUUUUAGAGCUA(SEQ ID NO:2404);(X17.18 or X17-19) GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:2407); or(X17-18 or X17-19)GUUUUAGAGCUAUGCU (SEQ ID NO :2408);(X17-18 orX17-19)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG(XN) (SEQ ID NO:1);(X17-18 orX17-19)GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCU AGUCCGUUAUC(Xn) (SEQ ID NO :2);(X17-18 orX1749)GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGU UAAAAUAAGGCUAGUCCGUUAUC(Xn) (SEQ ID NO:3);(X17-18)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCXn) (SEQ ID NO :4), (X17_18 orX17-19)GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(SEQ ID NO :5);(X17.18 orX17_i9)GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:6); or(X17.18 orX17.19)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:7);wherein X!7-18 0rX!749is a sequence (of 17-18 or 17-19 nucleotides) complementary to the complementary strand of a selected target sequence, preferably a target sequence immediately 5 ’ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or PCT/US2014/029068 WO 2014/144592 NNGG (see, for example, the configuration in Figure 1), and Xn is any sequence, wherein N (in the RNA) can be 0-200, e.g., 0-100, 0-50, or 0-20, that does not interfere with the binding of the ribonucleic acid to Cas9. In no case is the X!7-!8 or X17-9 identical to a sequence that naturally occurs adjacent to the rest of the RNA. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3’ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNAPolIII transcription. In some embodiments the RNAincludes one or more, e.g., up to 3, e.g., one, two, or three, additional nucleotides at the 5 ’ end of the RNA molecule that is not complementary to the target sequence. In some embodiments, the target complementarity region consists of 17-nucleotides (of target complementarity). In some embodiments, the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a selected target sequence, In some embodiments, the complementarity region is complementary to 18 consecutiveIn another aspect, the invention provides DNA molecules encoding the ribonucleic acids described herein, and host cells harboring or expressing the ribonucleic acids or vectors.In a further aspect, the invention provides methods for increasing specificity of RNA-guided genome editing in a cell, the method comprising contacting the cell with a guide RNA that includes a complementarity region consisting of 17-18 or 17-nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence, as described herein.In yet another aspect, the invention provides methods for inducing a single or double-stranded break in a target region of a double-stranded DNA molecule, e.g., in a genomic sequence in a cell. The methods include expressing in or introducing into the cell: a Cas9 nuclease or nickase; and a guide RNA that includes a sequence consisting of 17 or 18 or 19 nucleotides that are complementary to the complementary strand of a selected target sequence, preferably a target sequence immediately 5 ’ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG, e.g., a ribonucleic acid as described herein.Also provided herein are methods for modifying a target region of a double- stranded DNA molecule in a cell. The methods include expressing in or introducing into the cell: a dCas9-heterologous functional domain fusion protein (dCas9-HFD); PCT/US2014/029068 WO 2014/144592 and a guide RNAthat includes a complementarity region consisting of 17-18 or 17-nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence, as described herein.In some embodiments, the guide RNA is (i) a single guide RNA that includes a complementarity region consisting of 17-18 or 17-19 nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence, or (ii) a crRNAthat includes a complementarity region consisting of 17-18 or 17-19 nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA,In some embodiments, the target complementarity region consists of 17-nucleotides (of target complementarity). In some embodiments, the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a selected target sequence. In some embodiments, the complementarity region is complementary to 18 consecutiveIn no case is the X!7.!8 or X!7.!9 of any of the molecules described herein identical to a sequence that naturally occurs adj acent to the rest of the RNA. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3’ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA P01III transcription. In some embodiments the RNA includes one or more, e.g., up to 3, e.g., one, two, or three, additional nucleotides at the 5’ end of the RNA molecule that is not complementary to the target sequence.In some embodiments, one or more of the nucleotides of the RNA is modified, e.g., locked (2’-O-4’-C methylene bridge), is 5-methylcytidine, is 2'-O-methyl- pseudouridine, or in which the ribose phosphate backbone has been replaced by a polyamide chain, e.g., one or more of the nucleotides within or outside the target complementarity region X17.18 or X17.19. In some embodiments, some or all of the tracrRNA or crRNA, e.g., within or outside the X17-18 or X!7.!9 target complementarity region, comprises deoxyribonucleotides (e.g., is all or partially DNA, e.g. DNA/RNA hybrids).In an additional aspect, the invention provides methods for modifying a target region of a double-stranded DNA molecule, e.g., in a genomic sequence in a cell. The methods include expressing in or introducing into the cell: PCT/US2014/029068 WO 2014/144592 a dCas9-heterologous functional domain fusion protein (dCas9-HFD); anda guide RNA that includes a sequence consisting of 17-18 or 17-19 nucleotides that are complementary to the complementary strand of a selected target sequence, preferably a target sequence immediately 5’ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG, e.g., a ribonucleic acid as described herein. In no case is the X17-18 or X!7-19 identical to a sequence that naturally occurs adjacent to the rest of the RNA. In some embodiments the RNA includes one or more, e.g., up to 3, e.g., one, two, or three, additional nucleotides at the 5 ’ end of the RNA molecule that is not complementary to the target sequence.In another aspect, the invention provides methods for modifying, e.g., introducing a sequence specific break into, a target region of a double-stranded DNA molecule, e.g., in a genomic sequence in a cell. The methods include expressing in or introducing into the cell: a Cas9 nuclease or nickase, or a dCas9-heterologous functional domain fusion protein (dCas9-HFD);a tracrRNA, e.g., comprising or consisting of the sequenceGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO :8) or an active portion thereof;UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO:2405) or an active portion thereof;;AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC (SEQ ID NO:2407) or an active portion thereof;CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:2409) or an active portion thereof;UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG (SEQ ID NO:2410) or an active portion thereof;UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA (SEQ ID NO:2411) or an active portion thereof; orUAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO:2412) or an active portion thereof; anda crRNA that includes a sequence consisting of 17-18 or 17-19 nucleotides that are complementary to the complementary strand of a selected target sequence, preferably a target sequence immediately 5 ’ of a protospacer adjacent motif (PAM), e.g., NGG, PCT/US2014/029068 WO 2014/144592 NAG, or NNGG; in some embodiments the crRNA has the sequence: (X17-18 or X17-19)GUUUUAGAGCUA (SEQ ID NO:2404);(X17.18or X17-19) GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:2407); or (XI7.18 or X!7-19)GUUUUAGAGCUAUGCU (SEQ ID NO :2408).In some embodiments the crRNA is (X!7.18 or X!7. 19)GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:2407) and the tracrRNA is GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO :8); the cRNA is (X17-18 or X17.l9)GUUUUAGAGCUA (SEQ ID NO:2404) and the tracrRNA is UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO:2405); or the cRNA is (X!7.18 or Xi749) GUUUUAGAGCUAUGCU (SEQ ID NO :2408) and the tracrRNA is AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC (SEQ ID NO:2406).In no case is the X,7-18 or X]7.19 identical to a sequence that naturally occurs adjacent to the rest of the RNA. In some embodiments the RNA (e.g., tracrRNA or crRNA) includes one or more U, e.g., 2 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3’ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA P01III transcription. In some embodiments the RNA (e.g., tracrRNA or crRNA) includes one or more, e.g., up to 3, e.g., one, two, or three, additional nucleotides at the 5 ’ end of the RNA molecule that is not complementary to the target sequence. In some embodiments, one or more of the nucleotides of the crRNA or tracrRNA is modified, e.g., locked (2’-O-4’-C methylene bridge), is 5'- methylcytidine, is 2'-O-methyl-pseudouridine, or in which the ribose phosphate backbone has been replaced by a polyamide chain, e.g., one or more of the nucleotides within or outside the sequence X17-18 or X!7.19. In some embodiments, some or all of the tracrRNA or crRNA, e.g., within or outside the X!7.18 or X!7_target complementarity region, comprises deoxyribonucleotides (e.g., is all or partially DNA, e.g. DNA/RNA hybrids).In some embodiments, the dCas9-heterologous functional domain fusion protein (dCas9-HFD) comprises a HFD that modifies gene expression, histones, or DNA, e.g., transcriptional activation domain, transcriptional repressors (e.g., silencers such as Heterochromatin Protein 1 (HP1), e.g., HPla or HP), enzymes that modify PCT/US2014/029068 WO 2014/144592 the methylation state of DNA (e.g., DNA methyltransferase (DNMT) or TET proteins, e.g., TET1), or enzymes that modify histone subunit (e.g., histone acetyltransferases (HAT), histone deacetylases (HDAC), or histone demethylases). In preferred embodiments, the heterologous functional domain is a transcriptional activation domain, e.g., a VP64 or NF-kB p65 transcriptional activation domain; an enzyme that catalyzes DNA demethylation, e.g., a TET protein family member or the catalytic domain from one of these family members; or histone modification (e.g., ESDI, histone methyltransferase, HDACs, or HATs) or a transcription silencing domain, e.g., from Heterochromatin Protein 1 (HP1), e.g., HPla or HP Ip; or a biological tether, e.g., MS2, CRISPR/Cas Subtype Ypest protein 4 (Csy4) or lambda N protein. dCas9- HFD are described in a U.S. Provisional Patent Applications USSN 61/799,647, Filed on March 15, 2013, Attorney docket no, 00786-0882P02, USSN 61/838,148, filed on 6/21/2013, and PCT International Application No. PCT/US14/27335, all of which are incorporated herein by reference in its entirety.In some embodiments, the methods described herein result in an indel mutation or sequence alteration in the selected target genomic sequence.In some embodiments, the cell is a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS Figure 1: Schematic illustrating a gRNA/Cas9 nuclease complex bound to its target DNA site. Scissors indicate approximate cleavage points of the Cas9 nuclease PCT/US2014/029068 WO 2014/144592 on the genomic DNA target site. Note the numbering of nucleotides on the guide RNA proceeds in an inverse fashion from 5 ’ to 3 ’.Figure 2A: Schematic illustrating a rationale for truncating the 5 ’ complementarity region of a gRNA. Thick grey lines = target DNA site, thin dark grey line structure = gRNA, black lines show base pairing (or lack thereof) between gRNA and target DNA site.Figure 2B: Schematic overview of the EGFP disruption assay. Repair of targeted Cas9-mediatcd double-stranded breaks in a single integrated EGFP-PEST reporter gene by error-prone NHEJ-mediated repair leads to frame-shift mutations that disrupt the coding sequence and associated loss of fluorescence in cells.Figures 2C-F: Activities of RNA-guided nucleases (RGNs) harboring single guide RNAs (gRNAs) bearing (C) single mismatches, (D) adjacent double mismatches, (E) variably spaced double mismatches, and (F) increasing numbers of adjacent mismatches assayed on three different target sites in the EGFP reporter gene sequence. Mean activities of replicates are shown, normalized to the activity of a perfectly matched single gRNA. Error bars indicate standard errors of the mean. Positions mismatched in each single gRNA are highlighted in grey in the grid below. Sequences of the three EGFP target sites were as follows: EGFP Site 1 GGGCACGGGCAGCTTGCCGGTGG (SEQ ID NO:9) EGFP Site 2 GATGCCGTTCTTCTGCTTGTCGG (SEQ ID NO: 10) EGFP Site 3 GGTGGTGCAGATGAACTTCAGGG (SEQ IDNO:11) Figure 2G: Mismatches at the 5' end of the gRNA make CRISPR/Cas more sensitive more 3' mismatches. The gRNAs Watson-Crick base pair between the RNA&DNA with the exception of positions indicated with an "m" which are mismatched using the Watson-Crick transversion (i.e., EGFP Site#2 M18-19 is mismatched by changing the gRNA to its Watson-Crick partner at positions 18 & 19. Although positions near the 55 of the gRNA are generally very well tolerated, matches in these positions are important for nuclease activity when other residues are mismatched. When all four positions are mismatched, nuclease activity is no longer detectable. This further demonstrates that matches at these 5' position can help compensate for mismatches at other more 3' positions. Note these experiments were PCT/US2014/029068 WO 2014/144592 performed with a non-codon optimized version of Cas9 which can show lower absolute levels of nuclease activity as compared to the codon optimized version.Figure 2H: Efficiency of Cas9 nuclease activities directed by gRNAs bearing variable length complementarity regions ranging from 15 to 25 nts in a human cell- based U2OS EGFP disruption assay. Expression of a gRNA from the U6 promoter requires the presence of a 5 ’ G and therefore it was only possible to evaluate gRNAs harboring certain lengths of complementarity to the target DNA site (15, 17, 19, 20, 21, 23, and 25 nts).Figure 3 A: Efficiencies of EGFP disruption in human cells mediated by Casand full-length or shortened gRNAs for four target sites in the EGFP reporter gene. Lengths of complementarity regions and corresponding target DNA sites are shown. Ctrl = control gRNA lacking a complementarity region.Figure 3B: Efficiencies of targeted indel mutations introduced at seven different human endogenous gene targets by matched standard RGNs (Cas9 and standard full-length gRNAs) and tru-RGNs (Cas9 and gRNAs bearing truncations in their 5 ’ complementarity regions). Lengths of gRNA complementarity regions and corresponding target DNA sites are shown, Indel frequencies were measured by T7EI assay. Ctrl = control gRNA lacking a complementarity region.Figure 3C: DNA sequences of indel mutations induced by RGNs using a tru- gRNA or a matched full-length gRNA targeted to the EMX1 site. The portion of the target DNA site that interacts with the gRNA complementarity region is highlighted in grey with the first base of the PAM sequence shown in lowercase. Deletions are indicated by dashes highlighted in grey and insertions by italicized letters highlighted in grey. The net number of bases deleted or inserted and the number of times each sequence was isolated are shown to the right.Figure 3D: Efficiencies of precise HDR/SSODN-mediated alterations introduced at two endogenous human genes by matched standard and tru-RGNs. %HDR was measured using a BamHY restriction digest assay (see the Experimental Procedures for Example 2). Control gRNA = empty U6 promoter vector.Figure 3E: U2OS.EGFP cells were transfected with variable amounts of full- length gRNA expression plasmids (top) or tru-gRNA expression plasmids (bottom) together with a fixed amount of Cas9 expression plasmid and then assayed for percentage of cells with decreased EGFP expression. Mean values from duplicate experiments are shown with standard errors of the mean. Note that the data obtained PCT/US2014/029068 WO 2014/144592 with tru-gRNA matches closely with data from experiments performed with fall- length gRNA expression plasmids instead of tru-gRNA plasmids for these three EGFP target sites,Figure 3F: U2OS.EGFP cells were transfected with variable amount of Casexpression plasmid together with fixed amounts of full-length gRNA expression plasmids (top) or tru-gRNA expression plasmids (bottom) for each target (amounts determined for each tru-gRNA from the experiments of Figure 3E). Mean values from duplicate experiments are shown with standard errors of the mean. Note that the data obtained with tru-gRNA matches closely with data from experiments performed with full-length gRNA expression plasmids instead of tru-gRNA plasmids for these three EGFP target sites. The results of these titrations determined the concentrations of plasmids used in the EGFP disruption assays performed in Examples 1 and 2.Figure 4A: Schematic illustrating locations of VEGFA sites 1 and 4 targeted by gRNAs for paired double nicks. Target sites for the full-length gRNAs are underlined with the first base in the PAM sequence shown in lowercase. Location of the BamHI restriction site inserted by HDR with a ssODN donor is shown.Figure 4B: A tru-gRNA can be used with a paired nickase strategy to efficiently induce indel mutations. Substitution of a full-length gRNA for VEGFA site 1 with a tru-gRNA does not reduce the efficiency of indel mutations observed with a paired full-length gRNA for VEGFA site 4 and Cas9-D10A nickases. Control gRNA used is one lacking a complementarity region.Figure 4C: A tru-gRNA can be used with a paired nickase strategy to efficiently induce precise HDR/SSODN-mediated sequence alterations. Substitution of a full-length gRNA for VEGFA site 1 with a tru-gRNA does not reduce the efficiency of indel mutations observed with a paired full-length gRNA for VEGFA site 4 and Cas9-D10A nickases with an ssODN donor template. Control gRNA used is one lacking a complementarity region.Figure 5A: Activities of RGNs targeted to three sites in EGFP using full- length (top) or tru-gRNAs (bottom) with single mismatches at each position (except at the 5 ’-most base which must remain a G for efficient expression from the Upromoter) . Grey boxes in the grid below represent positions of the Watson-Crick transversion mismatches. Empty gRNA control used is a gRNA lacking a complementarity region. RGN activities were measured using the EGFP disruption assay and values shown represent the percentage of EGFP-ncgativc observed relative PCT/US2014/029068 WO 2014/144592 to an RGN using a perfectly matched gRNA. Experiments were performed in duplicate and means with error bars representing standard errors of the mean are shown.Figure 5B: Activities of RGNs targeted to three sites in EGFP using full- length (top) or tru-gRNAs (bottom) with adjacent double mismatches at each position (except at the 5’-most base which must remain a G for efficient expression from the U6 promoter). Data presented as in 5A.Figure 6A: Absolute frequencies of on- and off-target indcl mutations induced by RGNs targeted to three different endogenous human gene sites as measured by deep sequencing. Indel frequencies are shown for the three target sites from cells in which targeted RGNs with a full-length gRNA, a tru-gRNA, or a control gRNA lacking a complementarity region were expressed. Absolute counts of indel mutations used to make these graphs can be found in Table 3B.Figure 6B: Fold-improvements in off-target site specificities of three tru- RGNs. Values shown represent the ratio of on/off-target activities of tru-RGNs to on/off-target activities of standard RGNs for the off-target sites shown, calculated using the data from (A) and Table 3B. For the sites marked with an asterisk (*), no indels were observed with the tru-RGN and therefore the values shown represent conservative statistical estimates for the fold-improvements in specificities for these off-target sites (see Results and Experimental Procedures).Figure 6C, top: Comparison of the on-target and an off-target site identified by T7EI assay for the tru-RGN targeted to VEGFA site 1 (more were identified by deep sequencing). Note that the full-length gRNA is mismatched to the two nucleotides at the 5’ end of the target site and that these are the two nucleotides not present in the tru-gRNA target site. Mismatches in the off-target site relative to the on-target are highlighted in bold underlined text. Mismatches between the gRNAs and the off- target site are shown with X’s.Figure 6C, bottom: Indel mutation frequencies induced in the off-target site by RGNs bearing full-length or truncated gRNAs. Indel mutation frequencies were determined by T7EI assay. Note that the off-target site in this figure is one that we had examined previously for indel mutations induced by the standard RGN targeted to VEGFA site 1 and designated as site OT1-30 in that earlier study (Example 1 and Fu et al., NatBiotechnol. 31(9):822-6 (2013)). It is likely that we did not identify off- target mutations at this site in our previous experiments because the frequency of PCT/US2014/029068 WO 2014/144592 indel mutations appears to be at the reliable detection limit of the T7EI assay (2 - 5%).Figures 7A-D: DNA sequences of indel mutations induced by RGNs using tru-gRNAS or matched full-length gRNAs targeted to VEGFA sites 1 and 3. Sequences depicted as in Figure 3C.Figure 7E. Indel mutation frequencies induced by tru-gRNAs bearing a mismatched 5’ G nucleotide. Indel mutation frequencies in human U2OS.EGFP cells induced by Cas9 directed by tru-gRNAs bearing 17, 18 or 20 nt complementarity regions for VEGFA sites 1 and 3 and EMX1 site 1 are shown. Three of these gRNAs contain a mismatched 5 ’ G (indicated by positions marked in bold text). Bars indicate results from experiments using full-length gRNA (20 nt), tru-gRNA (17 or 18 nt), and tru-gRNA with a mismatched 5’ G nucleotide (17 or 18 nt with boldface T at 5’ end). (Note that no activity was detectable for the mismatched tru-gRNA to EMX1 site 1.)Figures 8A-C: Sequences of off-target indel mutations induced by RGNs in human U2OS.EGFP cells. Wild-type genomic off-target sites recognized by RGNs (including the PAM sequence) are highlighted in grey and numbered as in Table 1 and Table B. Note that the complementary strand is shown for some sites. Deleted bases are shown as dashes on a grey background. Inserted bases are italicized and highlighted in grey.Figures 9A-C: Sequences of off-target indel mutations induced by RGNs in human HEK293 cells. Wild-type genomic off-target sites recognized by RGNs (including the PAM sequence) are highlighted in grey and numbered as in Table 1 and Table B. Note that the complementary strand is shown for some sites. Deleted bases are shown as dashes on a grey background. Inserted bases are italicized and highlighted in grey. *Yielded a large number of single bp indels.

DETAILED DESCRIPTION CRISPR RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Although Marraffini and colleagues (Jiang et al.,Nat Biotechnol 31,233-239 (2013)) recently performed a systematic investigation of Cas9 RGN specificity in bacteria, the specificities of RGNs in human cells have not been extensively defined. Understanding the scope of RGN-mediated off-target effects in human and other eukaryotic cells will be critically essential if these nucleases are to be used widely for research and therapeutic applications. The present PCT/US2014/029068 WO 2014/144592 inventors have used a human cell-based reporter assay to characterize off-target cleavage of Cas9-based RGNs. Single and double mismatches were tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. Off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells were readily detected by examination of partially mismatched sites. The off-target sites identified harbor up to five mismatches and many are mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Thus RGNs arc highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.The results described herein reveal that predicting the specificity profile of any given RGN is neither simple nor straightforward. The EGFP reporter assay experiments show that single and double mismatches can have variable effects on RGN activity in human cells that do not strictly depend upon their position(s) within the target site. For example, consistent with previously published reports, alterations in the 3 ’ half of the sgRNA/DNA interface generally have greater effects than those in the 5’ half (Jiang et al., Nat Biotechnol 31, 233-239 (2013); Cong et al,, Science 339, 819-823 (2013); Jinek et al., Science 337, 816-821 (2012)); however, single and double mutations in the 3’ end sometimes also appear to be well tolerated whereas double mutations in the 5’ end can greatly diminish activities. In addition, the magnitude of these effects for mismatches at any given position(s) appears to be site- dependent. Comprehensive profiling of a large series of RGNs with testing of all possible nucleotide substitutions (beyond the Watson-Crick transversions used in our EGFP reporter experiments) may help provide additional insights into the range of potential off-targets. In this regard, the recently described bacterial cell-based method of Marraffini and colleagues (Jiang et al., Nat Biotechnol 31, 233-239 (2013)) or the in vitro, combinatorial library-based cleavage site-selection methodologies previously applied to ZFNs by Liu and colleagues (Pattanayak et al., Nat Methods 8, 765-7(2011)) might be useful for generating larger sets of RGN specificity profiles.Despite these challenges in comprehensively predicting RGN specificities, it was possible to identify bona fide off-targets of RGNs by examining a subset of genomic sites that differed from the on-target site by one to five mismatches. Notably, under conditions of these experiments, the frequencies of RGN-induced mutations at many of these off-target sites were similar to (or higher than) those observed at the PCT/US2014/029068 WO 2014/144592 intended on-target site, enabling the detection of mutations at these sites using the T7EI assay (which, as performed in our laboratory, has a reliable detection limit of ~to 5% mutation frequency). Because these mutation rates were very high, it was possible to avoid using deep sequencing methods previously required to detect muchlower frequency ZFN- and TALEN-induced off-target alterations (Pattanayak et al., Nat Methods 8, 765-770 (2011); Perez et al., Nat Biotechnol 26, 808-816 (2008); Gabriel et al., Nat Biotechnol 29, 816-823 (2011); Hockemeyer et al., Nat Biotechnol 29, 731-734 (2011)). Analysis of RGN off-target mutagenesis in human cells also confirmed the difficulties of predicting RGN specificities - not all single and double mismatched off-target sites show evidence of mutation whereas some sites with as many as five mismatches can also show alterations. Furthermore, the bona fide off- target sites identified do not exhibit any obvious bias toward transition or transversion differences relative to the intended target sequence (Table E;grey highlighted rows).Although off-target sites were seen for a number of RGNs, identification of these sites was neither comprehensive nor genome-wide in scale. For the six RGNs studied, only a very small subset of the much larger total number of potential off- target sequences in the human genome (sites that differ by three to six nucleotides from the intended target site; compare Tables E and C)was examined. Although examining such large numbers of loci for off-target mutations by T7EI assay isneither a practical nor a cost-effective strategy, the use of high-throughput sequencing in future studies might enable the interrogation of larger numbers of candidate off- target sites and provide a more sensitive method for detecting bona fide off-target mutations. For example, such an approach might enable the unveiling of additional off-target sites for the two RGNs for which we failed to uncover any off-targetmutations. In addition, an improved understanding both of RGN specificities and of any epigenomic factors (e.g., DNA methylation and chromatin status) that may influence RGN activities in cells might also reduce the number of potential sites that need to be examined and thereby make genome-wide assessments of RGN off-targets more practical and affordable.As described herein, a number of strategies can be used to minimize thefrequencies of genomic off-target mutations. For example, the specific choice of RGN target site can be optimized; given that off-target sites that differ at up to five positions from the intended target site can be efficiently mutated by RGNs, choosing target sites with minimal numbers of off-target sites as judged by mismatch counting PCT/US2014/029068 WO 2014/144592 seems u nlik ely to be effective; thousands of potential off-target sites that differ by four or five positions within the 20 bp RNA:DNA complementarity region will typically exist for any given RGN targeted to a sequence in the human genome (see, for example, Table C).It is also possible that the nucleotide content of the gRNA complementarity region might influence the range of potential off-target effects. For example, high GC-content has been shown to stabilize RNA:DNA hybrids (Sugimoto et al., Biochemistry 34, 11211-11216 (1995)) and therefore might also be expected to make gRNA/genomic DNA hybridization more stable and more tolerant to mismatches. Additional experiments with larger numbers of gRNAs will be needed to assess if and how these two parameters (numbers of mismatched sites in the genome and stability of the RNA:DNA hybrid) influence the genome-wide specificities of RGNs. However, it is important to note that even if such predictive parameters can be defined, the effect of implementing such guidelines would be to further restrict the targeting range of RGNs.One potential general strategy for reducing RGN-induced off-target effects might be to reduce the concentrations of gRNA and Cas9 nuclease expressed in the cell. This idea was tested using the RGNs for VEGFA target sites 2 and 3 in U2OS.EGFP cells; transfecting less sgRNA- and Cas9-expressing plasmid decreased the mutation rate at the on-target site but did not appreciably change the relative rates of off-target mutations (Tables 2A and 2B).Consistent with this, high-level off-target mutagenesis rates were also observed in two other human cell types (HEK293 and K562 cells) even though the absolute rates of on-target mutagenesis are lower than in U2OS.EGFP cells. Thus, reducing expression levels of gRNA and Cas9 in cells is not likely to provide a solution for reducing off-target effects. Furthermore, these results also suggest that the high rates of off-target mutagenesis observed in human cells are not caused by overexpression of gRNA and/or Cas9.

Table 2A Indel mutation frequencies at on- and off-target genomic sites induced by different amounts of Cas9- and single gRNA-expressing plasmids 5 ____________________ for the RGN targeted to VEGFA Target Site 2_____ ______ Site Sequence SEQ ID NO: 250ng gRNA/750 ng Cas9 Mean indel frequency (%) ± SEM 12.5ng gRNA/50 ng Cas9 Mean indel frequency (%) ± SEM T2 (On-target) GACCCCCTCCACCCCGCCTCCGG 50.2 ±4.9 25.4 ±4.8 OT2-1 GACCCCCCCCACCCCGCCCCCGG 14.4 ±3.4 4.2 ±0.2 OT2-2 GGGCCCCTCCACCCCGCCTCTGG 20.0 ±6.2 9.8 ±1.1 OT2-6 CTACCCCTCCACCCCGCCTCCGG 8.2 ±1.4 6.0 ±0.5 OT2-9 GCCCCCACCCACCCCGCCTCTGG 50.7 ±5.6 16.4 ± 2.1 OT2-15 TACCCCCCACACCCCGCCTCTGG 9.7 ±4.5 2.1 ±0.0 OT2-17 ACACCCCCCCACCCCGCCTCAGG 14.0 ± 2.8 7.1 ±0.0 OT2-19 ATTCCCCCCCACCCCGCCTCAGG 17.0 ±3.3 9.2 ±0.4 OT2-20 CCCCACCCCCACCCCGCCTCAGG 6.1 ±1.3 N.D. OT2-23 CGCCCTCCCCACCCCGCCTCCGG 44.4 ±6.7 35.1 ± 1.8 OT2-24 CTCCCCACCCACCCCGCCTCAGG 62.8 ±5.0 44.1 ±4.5 OT2-29 TGCCCCTCCCACCCCGCCTCTGG 13.8 ±5.2 5.0 ±0.2 OT2-34 AGGCCCCCACACCCCGCCTCAGG 2.8 ± 1.5 N.D. Amounts of gRNA- and Cas9-expressing plasmids transfected into U2OS.EGFP cells for these assays are shown at the top of each column. (Note that data for 250 ng gRNA/750 ng Cas9 are the same as those presented in Table1.) Mean indel frequencies were determined using the T7EI assay from replicate samples as described in Methods.OT = Off-target sites, numbered as in Table1 and Table B. Mismatches from the on-target site (within the 20 bp region to which the gRNA hybridizes) are highlighted as bold, underlined text. N.D. = none detected W O 2014/144592 PCT/US2014/029068 Table 2B Indel mutation frequencies at on- and off-target genomic sites induced by different amounts of Cas9- and single gRNA-expressing plasmids for the RGN ______ targeted to VEGFA Target Site 3___________________ Site Sequence SEQ ID NO: 250ng gRNA/750 ng Cas9 Mean indel frequency (%)±SEM 12.5ng gRNA/250 ng Cas9 Mean indel frequency (%) ± SEM T3 (On-target) GGTGAGTGAGTGTGTGCGTGTGG 49.4 ± 3.8 33.0 ±3.7 OT3-1 GGTGAGTGAGTGTGTGTGTGAGG 7.4 ±3.4 N.D. OT3-2 AGTGAGTGAGTGTGTGTGTGGGG 24.3 ±9.2 9.8 ±4.2 OT3-4 GCTGAGTGAGTGTATGCGTGTGG 20.9 ±11.8 4.2 ± 1.2 OT3-9 GGTGAGTGAGTGCGTGCGGGTGG 3.2 ±0.3 N.D. OT3-17 GTTGAGTGAATGTGTGCGTGAGG 2.9 ±0.2 N.D. OT3-18 TGTGGGTGAGTGTGTGCGTGAGG 13.4 ± 4.2 4.9 ± 0.0 OT3-2O AGAGAGT GAGTGT GT GCATGAGG 16.7 ±3.5 7.9 ±2.4 Amounts of gRNA- and Cas9-expressing plasmids transfected into U2OS.EGFP cells for these assays are shown at the top of each column. (Note that data for 250 ng gRNA/750 ng Cas9 are the same as those presented in Table 1.) Mean indel frequencies were determined using the T7EI assay from replicate samples as described in Methods . OT = Off-target sites, numbered as in Table 1 and Table B. N.D. = none detected W O 2014/144592 dO PC T/U S2014/029068 PCT/US2014/029068 WO 2014/144592 The finding that significant off-target mutagenesis can be induced by RGNs in three different human cell types has important implications for broader use of this genome-editing platform. For research applications, the potentially confounding effects of high frequency off-target mutations will need to be considered, particularly for experiments involving either cultured cells or organisms with slow generation times for which the outcrossing of undesired alterations would be challenging. One way to control for such effects might be to utilize multiple RGNs targeted to different DNA sequences to induce the same genomic alteration because off-target effects are not random but instead related to the targeted site. However, for therapeutic applications, these findings clearly indicate that the specificities of RGNs will need to be carefully defined and/or improved if these nucleases are to be used safely in the longer term for treatment of human diseases.

Methods for Improving Specificity As shown herein, CRISPR-Cas RNA-guided nucleases based on the S. pyogenes Cas9 protein can have significant off-target mutagenic effects that are comparable to or higher than the intended on-target activity (Example 1). Such off- target effects can be problematic for research and in particular for potential therapeutic applications. Therefore, methods for improving the specificity of CRISPR-Cas RNA guided nucleases (RGNs) are needed.As described in Example 1, Cas9 RGNs can induce high-frequency indel mutations at off-target sites in human cells (see also Cradick et al., 2013; Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013). These undesired alterations can occur at genomic sequences that differ by as many as five mismatches from the intended on- target site (see Example 1). In addition, although mismatches at the 5’ end of the gRNA complementarity region are generally better tolerated than those at the 3 ’ end, these associations are not absolute and show site-to-site-dependence (see Example and Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013). As a result, computational methods that rely on the number and/or positions of mismatches currently have limited predictive value for identifying bona fide off-target sites. Therefore, methods for reducing the frequencies of off-target mutations remain an important priority if RNA-guided nucleases are to be used for research and therapeutic applications.

PCT/US2014/029068 WO 2014/144592 Truncated Guide RNAs (tru-gRNAs) Achieve Greater Specificity Guide RNAs generally speaking come in two different systems: System 1, which uses separate crRNA and tracrRNAs that function together to guide cleavage by Cas9, and System 2, which uses a chimeric crRNA-tracrRNA hybrid that combines the two separate guide RNAs in a single system (referred to as a single guide RNA or sgRNA, see also Jinek et al., Science 2012; 337:816-821). The tracrRNA can be variably truncated and a range of lengths has been shown to function in both the separate system (system 1) and the chimeric gRNA system (system 2). For example, in some embodiments, tracrRNA may be truncated from its 3’ end by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts. In some embodiments, the tracrRNA molecule maybe truncated from its 5’ end by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts. Alternatively, the tracrRNAmolecule may be truncated from both the 5’ and 3’ end, e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 nts on the 5’ end and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts on the 3’ end. See, e.g., Jinek et al., Science 2012; 337:816-821; Mali et al., Science. 2013 Feb 15;339(6121):823-6; Cong et al ״ Science. 2013 Feb 15;339(6121):819-23; and Hwang and Fu et al., Nat Biotechnol. 2013 Mar;31(3):227-9; Jinek et al.. Elife 2, 600471 (2013)). For System 2, generally the longer length chimeric gRNAs have shown greater on-target activity but the relative specificities of the various length gRNAs currently remain undefined and therefore it may be desirable in certain instances to use shorter gRNAs. In some embodiments, the gRNAs are complementary to a region that is within about 100-800 bp upstream of the transcription start site, e.g., is within about 500 bp upstream of the transcription start site, includes the transcription start site, or within about 100-800 bp, e.g., within about 500 bp, downstream of the transcription start site. In some embodiments, vectors (e.g., plasmids) encoding more than one gRNA are used, e.g., plasmids encoding, 2, 3, 4, 5, or more gRNAs directed to different sites in the same region of the target gene.The present application describes a strategy for improving RGN specificity based on the seemingly counterintuitive idea of shortening, rather than lengthening, the gRNA complementarity region. These shorter gRNAs can induce various types of Cas9-mediated on-target genome editing events with efficiencies comparable to (or, in some cases, higher than) full-length gRNAs at multiple sites in a single integrated EGFP reporter gene and in endogenous human genes. In addition, RGNs using these shortened gRNAs exhibit increased sensitivity to small numbers of mismatches at the PCT/US2014/029068 WO 2014/144592 gRNA-target DMA interface. Most importantly, use of shortened gRNAs substantially reduces the rates of genomic off-target effects in human cells, yielding improvements of specificity as high as 5000-fold or more at these sites. Thus, this shortened gRNA strategy provides a highly effective approach for reducing off-target effects without compromising on-target activity and without the need for expression of a second, potentially mutagenic gRNA. This approach can be implemented on its own or in conjunction with other strategies such as the paired nickase method to reduce the off-target effects of RGNs in human cells.Thus, one method to enhance specificity of CRISPR/Cas nucleases shortens the length of the guide RNA (gRNA) species used to direct nuclease specificity. Casnuclease can be guided to specific 17-18 nt genomic targets bearing an additional proximal protospacer adjacent motif (PAM), e.g., of sequence NGG, using a guide RNA, e.g., a single gRNA or a crRNA (paired with a tracrRNA), bearing 17 or 18 nts at its 5’ end that are complementary to the complementary strand of the genomic DNA target site (Figure 1). Although one might expect that increasing the length of the gRNA complementarity region would improve specificity, the present inventors (Hwang et al., PL0S One. 2013 Jul 9;8(7):e68708) and others (Ran et al., Cell. 2013 Sep 12;154(6): 1380-9) have previously observed that lengthening the target site complementarity region at the 5 ’ end of the gRNA actually makes it function less efficiently at the on-target site.By contrast, experiments in Example 1 showed that gRNAs bearing multiple mismatches within a standard length 5 ’ complementarity targeting region could still induce robust Cas9-mediated cleavage of their target sites. Thus, it was possible that truncated gRNAs lacking these 5’-end nucleotides might show activities comparable to their full-length counterparts (Fig. 2A). It was further speculated that these 5 ’ nucleotides might normally compensate for mismatches at other positions along the gRNA-target DNA interface and therefore predicted that shorter gRNAs might be more sensitive to mismatches and thus induce lower levels of off-target mutations (Fig. 2A).Decreasing the length of the DNA sequence targeted might also decrease the stability of the gRNA:DNA hybrid, making it less tolerant of mismatches and thereby making the targeting more specific. That is, truncating the gRNA sequence to recognize a shorter DNA target might actually result in a RNA-guided nuclease that is PCT/US2014/029068 WO 2014/144592 less tolerant to even single nucleotide mismatches and is therefore more specific and has fewer unintended off-target effects.This strategy for shortening the gRNA complementarity region could potentially be used with RNA guided proteins other than S. pyogenes Cas9 including other Cas proteins from bacteria or archaea as well as Cas9 variants that nick a single strand of DNA or have no-nuclease activity such as a dCas9 bearing catalytic inactivating mutations in one or both nuclease domains. This strategy can be applied to systems that utilize a single gRNA as well as those that use dual gRNAs (e.g., the crRNA and tracrRNA found in naturally occurring systems).Thus, described herein is a single guide RNA comprising a crRNA fused to a normally trans-encoded tracrRNA, e.g., a single Cas9 guide RNA as described in Mali et al,, Science 2013 Feb 15; 339(6121):823-6, but with a sequence at the 5’ end that is complementary to fewer than 20 nucleotides (nts), e.g., 19,18, or 17 nts, preferably or 18 nts, of the complementary strand to a target sequence immediately 5’ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG. In some embodiments, the shortened Cas9 guide RNA consists of the sequence: (X17.18 or X!7-19)GUUUUAGAGCUA (SEQ ID NO:2404);(X17-18 or X17_19) GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:2407); or (Xi7.18 or X17-19)GUUUUAGAGCUAUGCU (SEQ ID NO:2408);(X17.18 orXI7_I9)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG(X N) (SEQ ID NO:1);(Xn-18 orX17.19)GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCU AGUCCGUUAUC(Xn) (SEQ ID NO :2);(X!7-18 orX17-19)GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGU UAAAAUAAGGCUAGUCCGUUAUC(Xn) (SEQ IDNO:3); (X17-18 or X17_19)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(Xn) (SEQ ID NO :4), (X17-18 orX17-19)GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(SEQ ID NO:5);(X17.18 or PCT/US2014/029068 WO 2014/144592 X17-19)GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:6);or (X!7-18 0rX17-19)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:7); wherein X!7.18 or X1749 is the nucleotide sequence complementary to 17-18 or 17-19 consecutive nucleotides of the target sequence, respectively. Also described herein are DNAs encoding the shortened Cas9 guide KN As that have been described previously in the literature (Jinek et al., Science. 337(6096):816-21 (2012) and Jinek et al., Elife. 2x00471 (2013)).The guide RNAS can include Xn which can be any sequence, wherein N (in the RNA) can be 0-200, e.g., 0-100, 0-50, or 0-20, that does not interfere with the binding of the ribonucleic acid to Cas9.In some embodiments, the guide RNA includes one or more Adenine (A) or Uracil (U) nucleotides on the 3 ’ end. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g,, U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3 ’ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA P01III transcription.Modified RNA oligonucleotides such as locked nucleic acids (LNAs) have been demonstrated to increase the specificity of RNA-DNA hybridization by locking the modified oligonucleotides in a more favorable (stable) conformation. For example, 2’-O-mcthyl RNA is a modified base where there is an additional covalent link age between the 2’ oxygen and 4’ carbon which when incorporated into oligonucleotides can improve overall thermal stability arid selectivity (formula I). formula I - Locked Nucleic AcidThus in some embodiments, the tru-gRNAs disclosed herein may comprise one or more modified RNA oligonucleotides. For example, the truncated guide RNAs molecules described herein can have one, some or all of the 17-18 or 17-19 nts 5’ PCT/US2014/029068 WO 2014/144592 region of the guideRNA complementary to the target sequence are modified, c.g., locked (2’-O-4’-C methylene bridge), 5'-methylcytidine, 2'-O-methyl-pseudouridine, or in which the ribose phosphate backbone has been replaced by a polyamide chain (peptide nucleic acid), e.g., a synthetic ribonucleic acid.In other embodiments, one, some or all of the nucleotides of the tru-gRNA sequence may be modified, e.g., locked (2’-O-4’-C methylene bridge), 5'- methylcytidine, 2'-O-methyl-pseudouridine, or in which the ribose phosphate backbone has been replaced by a polyamide chain (peptide nucleic acid), c.g., a synthetic ribonucleic acid.In a cellular context, complexes of Cas9 with these synthetic gRNAs could be used to improve the genome-wide specificity of the CRISPR/Cas9 nuclease system.Exemplary modified or synthetic tru-gRNAs may comprise, or consist of, the following sequences:(X17_18 or X17-19)GUUUUAGAGCUA(Xn) (SEQ ID NO:2404);(X17-18 or X17-19) GUUUUAGAGCUAUGCUGUUUUG (XN) (SEQ ID NO:2407); (X,7-18 or X17.19)GUUUUAGAGCUAUGCU(XN) (SEQ ID NO:2408);(X!7-18 orX17.19)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG(XN) (SEQ ID NO:1);(X17-18 orX17_19)GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCU AGUCCGUUAUC(Xn) (SEQ ID NO:2);(X17-18 orX1749)GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGU UAAAAUAAGGCUAGUCCGUUAUC(Xn) (SEQ ID NOG);(X17-18)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(XN) (SEQ ID NO :4), (X17-18 orX17_19)GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(SEQ ID NOG);(X17-18 orX17.19)GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:6); or PCT/US2014/029068 WO 2014/144592 (X17.18 orX17-19)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:7);wherein X!7-18 or X17-19 is a sequence complementary to 17-18 or 17-19 nts of a target sequence, respectively, preferably a target sequence immediately 5’ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG, and further wherein one or more of the nucleotides arc locked, e.g., one or more of the nucleotides within the sequence Xu-18 or Xu-19, one or more of the nucleotides within the sequence Xn, or one or more of the nucleotides within any sequence of the tru-gRNA. Xn is any sequence, wherein N (in the RNA) can be 0-200, e.g., 0-100, 0-50, or 0-20, that does not interfere with the binding of the ribonucleic acid to Cas9. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3 ’ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA P01III transcription.Although some of the examples described herein utilize a single gRNA, the methods can also be used with dual gRNAs (e.g., the crRNA and tracrRNA found in naturally occurring systems). In this case, a single tracrRNA would be used in conjunction with multiple different crRNAs expressed using the present system, e.g., the following: (Xu-18 or Xm9)GUUUUAGAGCUA (SEQ ID NO:2404);(Xu-18 or Xu-19) GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:2407); or (Xu-18 or Xu-19)GUUUUAGAGCUAUGCU (SEQ ID NO:2408); and a tracrRNA sequence. In this case, the crRNA is used as the guide RNA in the methods and molecules described herein, and the tracrRNA can be expressed from the same or a different DNA molecule. In some embodiments, the methods include contacting the cell with a tracrRNA comprising or consisting of the sequence GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO :8) or an active portion thereof (an active portion is one that retains the ability to form complexes with Cas9 or dCas9). In some embodiments, the tracrRNA molecule may be truncated from its 3’ end by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35 or 40 nts. In another embodiment, the tracrRNA molecule may be truncated from its 5 ’ end by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts. Alternatively, the PCT/US2014/029068 WO 2014/144592 tracrRNA molecule may be truncated from both, the 5’ and 3’ end, e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15 or 20 nts on the 5’ end and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts on the 3’ end. Exemplary tracrRNA sequences in addition to SEQ ID NO:8 include the following:UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO:2405) or an active portion thereof;AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC (SEQ ID NO:2407) or an active portion thereof;CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGGCACCGAGUCGGUGC (SEQ ID NO :2409) or an active portion thereof;UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG (SEQ ID NO:2410) or an active portion thereof;UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA (SEQ ID NO:2411) or an active portion thereof; or UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO:2412) or an active portion thereof.In some embodiments wherein (X!7,!8 or X!7. 19)GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:2407) is used as a crRNA, the following tracrRNA is used:GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO :8) or an active portion thereof. In some embodiments wherein (X!7-!8 or X!7-19)GUUUUAGAGCUA (SEQ ID NO:2404) is used as a crRNA, the following tracrRNA is used:UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO:2405) or an active portion thereof. In some embodiments wherein (X!7-18 or XI7-19) GUUUUAGAGCUAUGCU (SEQ ID NO:2408) is used as a crRNA, the following tracrRNA is used:AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC (SEQ ID NO:2406) or an active portion thereof.In addition, in a system that uses separate crRNA and tracrRNA, one or both can be synthetic and include one or more modified (e.g., locked) nucleotides or deoxyribonucleotides .

PCT/US2014/029068 WO 2014/144592 In some embodiments, the single guide RNAs and/or crRNAs and/or tracrRNAs can include one or more Adenine (A) or Uracil (U) nucleotides on the 3 ’ end.Existing Cas9-based RGNs use gRNA-DNA heteroduplex formation to guide targeting to genomic sites of interest. However, RNA-DNA heteroduplexes can form a more promiscuous range of structures than their DNA-DNA counterparts. In effect, DNA-DNA duplexes are more sensitive to mismatches, suggesting that a DNA- guided nuclease may not bind as readily to off-target sequences, making them comparatively more specific than RNA-guided nucleases. Thus, the truncated guide RNAs described herein can be hybrids, i.e., wherein one or more deoxyribonucleotides, e.g., a short DNA oligonucleotide, replaces all or part of the gRNA, e.g., all or part of the complementarity region of a gRNA, This DNA-based molecule could replace either all or part of the gRNA in a single gRNA system or alternatively might replace all of part of the crRNA in a dual crRNA/tracrRNA system. Such a system that incorporates DNA into the complementarity region should more reliably target the intended genomic DNA sequences due to the general intolerance of DNA-DNA duplexes to mismatching compared to RNA-DNA duplexes. Methods for making such duplexes are known in the art, See, e.g., Barker et al., BMC Genomics. 2005 Apr 22;6:57; and Sugimoto et al., Biochemistry. 20Sep 19;39(37): 11270-81.Exemplary modified or synthetic tru-gRNAs may comprise, or consist of, the following sequences:(X17-18 orX17-19)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG(XN) (SEQ ID NO:1);(X!7-18 orX17-19)GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCU AGUCCGUUAUC(Xn) (SEQ ID NO :2);(X!7-18 orX17u9)GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGU UAAAAUAAGGCUAGUCCGUUAUC(Xn) (SEQ ID NO:3); (X!7-18 or X17-19)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(Xn) (SEQ ID NO:4), (X17.18 or PCT/US2014/029068 WO 2014/144592 X17-19)GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(SEQ ID NO :5);(X17-18 orX17.19)GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO :6); or (X17-18 orX17_19)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:7);wherein X17-18 or X!7_19 is a sequence complementary to 17-18 or 17-19 nts of a target sequence, respectively, preferably a target sequence immediately 5 ’ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG, and further wherein one or more ofthe nucleotides are deoxyribonucleotides, e.g., one ormore of the nucleotides within the sequence X17-18 or X!7.19, one or more of the nucleotides within the sequence Xn, or one or more of the nucleotides within any sequence of the tru-gRNA. Xn is any sequence, wherein N (in the RNA) can be 0-200, e.g., 0-100, 0-50, or 0-20, that does not interfere with the binding of the ribonucleic acid to Cas9. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3’ end ofthe molecule, as a result ofthe optional presence of one or more Ts used as a termination signal to terminate RNA P01III transcription.In addition, in a system that uses separate crRNA and tracrRNA, one or both can be synthetic and include one or more deoxyribonucleotides.In some embodiments, the single guide RNAs or crRNAs or tracrRNAs includes one or more Adenine (A) or Uracil (U) nucleotides on the 3 ’ end.In some embodiments, the gRNA is targeted to a site that is at least three or more mismatches different from any sequence in the rest of the genome in order to minimize off-target effects.The methods described can include expressing in a cell, or contacting the cell with, a shortened Cas9 gRNA (tru-gRNA) as described herein (optionally a modified or DNA/RNA hybrid tru-gRNA), plus a nuclease that can be guided by the shortened Cas9 gRNAs, e.g., a Cas9 nuclease, e.g., as described in Mali et al., a Cas9 nickase as PCT/US2014/029068 WO 2014/144592 described in Jinek et al., 2012; or a dCas9-hctcrofunctional domain fusion (dCas9- HFD).

Cas9 A number of bacteria express Cas9 protein variants. The Cas9 from Streptococcus pyogenes is presently the most commonly used; some of the other Casproteins have high levels of sequence identity with the S. pyogenes Cas9 and use the same guide RNAs. Others arc more diverse, use different gRNAs, and recognize different PAM sequences as well (the 2-5 nucleotide sequence specified by the protein which is adjacent to the sequence specified by the RNA). Chylinski et al. classified Cas9 proteins from a large group of bacteria (RNA Biology 10:5, 1-12; 2013), and a large number of Cas9 proteins are listed in supplementary figure 1 and supplementary table 1 thereof, which are incorporated by reference herein. Additional Cas9 proteins are described in Esvelt et al,, Nat Methods. 2013 Nov; 10(1 !):1116-21 and Fonfara et al., "Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Casamong orthologous type II CRISPR-Cas systems. " Nucleic Acids Res. 2013 Nov 22. [Epub ahead of print] doi:10.1093/nar/gktl074.Cas9 molecules of a variety of species can be used in the methods and compositions described herein. While the S. pyogenes and S. thermophilus Casmolecules are the subject of much of the disclosure herein, Cas9 molecules of, derived from, or based on the Cas9 proteins of other species listed herein can be used as well. In other words, while the much of the description herein uses S. pyogenes and 5. thermophilus Cas9 molecules, Cas9 molecules from the other species can replace them, Such species include those set forth in the following table, which was created based on supplementary figure 1 of Chylinski et al., 2013. Alternative Cas9 proteins GenBank AccNo. Bacterium 303229466 Veillonella atypica ACS-134-V-C0l7a34762592 Fusobacterium nucleatum subsp. vincentii374307738 Filifactor alocis ATCC 35896320528778 Solobacterium moorei F0204291520705 Coprococcus catus GD-742525843 Treponema denticola ATCC 35405304438954 Peptoniphilus duerdenii ATCC BAA-1640224543312 Catenibacterium mitsuokai DSM1589724379809 Streptococcus mutans UA15915675041 Streptococcus pyogenes SF37016801805 Listeria innocua Clipll262 PCT/US2014/029068 WO 2014/144592 Alternative Cas9 proteins GenBank Acc No. Bacterium116628213 Streptococcus thermophilus LMD-9323463801 Staphylococcus pseudintermedius ED99352684361 Acidaminococcus intestini RyC-MR95302336020 Olsenellauli DSM 7084366983953 Oenococcus kitaharae DSM17330310286728 Bifidobacterium bifidum SI 7258509199 Lactobacillus rhamnosus GG300361537 Lactobacillus gasseri JV-V03169823755 Finegoldia magna ATCC 2932847458868 Mycoplasma mobile 163K284931710 Mycoplasma gallisepticum str. F363542550 Mycoplasma ovipneumoniae SC01384393286 Mycoplasma cams PG 1471894592 Mycoplasma synoviae 53238924075 Eubacterium rectale ATCC 33656116627542 Streptococcus thermophilus LMD-9315149830 Enterococcus faecalis TX0012315659848 Staphylococcus lugdunensis M23590160915782 Eubacterium dolichum DSM 3991336393381 Lactobacillus coryniformis subsp. torquens310780384 Ilyobacter polytropus DSM 2926325677756 Ruminococcus albus 8187736489 Akkermansia muciniphila A TCC BAA-835117929158 Acidothermus cellulolyticus 11B189440764 Bifidobacterium longum DJO10A283456135 Bifidobacterium dentium Bdl38232678 Corynebacterium diphtheriae NCTC13129187250660 Elusimicrobium minutum Peil91319957206 Nitratifractor salsuginis DSM 16511325972003 Sphaerochaeta globus str. Buddy261414553 Fibrobacter succinogenes subsp. succinogenes60683389 Bacteroides fragilis NCTC 9343256819408 Capnocytophaga ochracea DSM 727190425961 Rhodopseudomonas palustris BisBl8373501184 Prevotella micans F0438294674019 Prevotella ruminicola 23365959402 Flavobacterium columnare ATCC 49512312879015 Aminomonas paucivorans DSM 1226083591793 Rhodospirillum rubrum A TCC 11170294086111 Candidatus Puniceispirillum marinum IMCC1322121608211 Verminephrobacter eiseniae EF01-2344171927 Ralstonia syzygii R24159042956 Dinoroseobacter shibae DFL 12288957741 Azospirillum sp- B51092109262 Nitrobacter hamburgensis XI4148255343 Bradyrhizobium sp- BTAil WO 2014/144592 PCT/US2014/029068 Alternative Cas9 proteins GenBank Acc No. Bacterium 34557790 Wolinella succinogenes DSM1740218563121 Campylobacter jejuni subsp. jejuni291276265 Helicobacter mustelae 12198229113166 Bacillus cereus Rockl-15222109285 Acidovorax ebreus TPSY189485225 uncultured Termite group 1182624245 Clostridium perfringens D str.220930482 Clostridium cellulolyticum Hl 0154250555 Pannbaculum lavamentivorans DS-1257413184 Roseburia intestinalis Ll-82218767588 Neisseria meningitidis Z249115602992 Pasteurella multocida subsp. multocida319941583 Sutterella wadsworthensis 3 1254447899 gamma proteobacterium HTCC501554296138 Legionella pneumophila str. Paris331001027 Parasutterella excrementihominis YIT1185934557932 Wolinella succinogenes DSM 1740118497352 Francisella novicida U112 The constructs and methods described herein can include the use of any of those Casproteins, and their corresponding guide RNAs or other guide RNAs that are compatible. The Cas9 from Streptococcus thennophilus LMD-9 CRISPRI system has also been shown to function in human cells in Cong et al (Science 339, 819(2013)). Cas9 orthologs from TV. meningitides are described in Hou et al,, Proc NatlAcad SciU S A. 2013 Sep 24;U0(39):15644-9 andEsvelt et al., Nat Methods. 20Nov;10(ll): 1116-21, Additionally, Jinek et al. showed in vitro that Cas9 orthologs from 5. thermophilus and L. innocua, (but not from N. meningitidis or C. jejuni, which likely use a different guide RNA), can be guided by a dual S. pyogenes gRNA tocleave target plasmid DNA, albeit with slightly decreased efficiency.In some embodiments, the present system utilizes the Cas9 protein fromS. pyogenes, either as encoded in bacteria or codon-optimized for expression in mammalian cells, containing mutations at DI 0, E762, H983, or D986 and H840 or N863, e.g., D10A/D10N and H840A/H840N/H840Y, to render the nuclease portion of the protein catalytically inactive; substitutions at these positions could be alanine (as they are in Nishimasu al,, Cell 156, 935-949 (2014)) or they could be other residues, e.g., glutamine, asparagine, tyrosine, serine, or aspartate, e.g.״ E762Q, H983N, H983Y, D986N, N863D, N863S, 01־N863H (Figure IC). The sequence of the catalytically inactive S. pyogenes Cas9 that can be used in the methods and PCT/US2014/029068 WO 2014/144592 compositions described herein is as follows; the exemplary mutations of D10A and H840A are in bold and underlined. 5MDKKYSIGLAATRLKRTARR 20IGTNSVGWAVRYTRRKNRIC 30ITDEYKVPSKYLQEIFSNEM 40KFKVLGNTDR100AKVDDSFFHR 50HSIKKNLIGA110LEESFLVEED 60LLFDSGETAE120KKHERHPIFG 10130NIVDEVAYHE140KYPTIYHLRK150KLVDSTDKAD160LRLIYLALAH170MIKFRGHFLI180EGDLNPDNSD190VDKLFIQLVQ200TYNQLFEENP210INASGVDAKA220ILSARLSKSR230RLENLIAQLP240GEKKNGLFGN250LIALSLGLTP260NFKSNFDLAE270DAKLQLSKDT280YDDDLDNLLA290QIGDQYADLF300LAAKNLSDAI 20310LLSDILRVNT370GYIDGGASQE 320EITKAPLSAS380EFYKFIKPII 330MIKRYDEHHQ390EKMDGTEELL 340DLTLLKALVR400VKLNREDLLR 350Q0LPEKYKEI410KORTFDNGSI 360FFDOSKNGYA420PHQIHLGELH 25430AILRROEDFY440PFLKDNREKI450EKILTFRIPY460YVGPLARGNS470RFAWMTRKSE480ETITPWNFEE490VVDKGASAQS500FIERMTNFDK510NLPNEKVLPK520HSLLYEYFTV530YNELTKVKYV540TEGMRKPAFL550SGEQKKAIVD560LLFKTNRKVT570VKQLKEDYFK580KIECFDSVEI590SGVEDRFNAS600LGTYHDLLKI 35610IKDKDFLDNE670RLSRKLINGI 620ENEDILEDIV680RDKQSGKTIL 6LTLTLFEDRE690DFLKSDGFAN 640MIEERLKTYA700RNFMQLIHDD 650HLFDDKVMKQ710SLTFKEDIQK 660LKRRRYTGWG720AQVSGQGDSL 40730HEHIANLAGS740PAIKKGILQT750VKVVDELVKV760MGRHKPENIV770IEMARENQTT780QKGOKNSRER790MKRIEEGIKE800LGSQILKEHP810VENTQLQNEK820LYLYYLONGR830DMYVDQELDI840NRLSDYDVDA850IVPQSFLKDD860SIDNKVLTRS870DKNRGKSDNV880PSEEVVKKMK890NYWRQLLNAK900LITQRKFDNL 50910TKAERGGLSE970KLVSDFRKDF 920LDKAGFIKRQ980QFYKVREINN 930LVETRQITKH990YHHAHDAYLN 940VAQILDSRMN1000AVVGTALIKK 950TKYDENDKLI1010YPKLESEFVY 960REVKVITLKS1020GDYKVYDVRK 551030MIAKSEQEIG1040KATAKYFFYS1050NIMNFFKTEI1060TLANGEIRKR1070PLIETNGETG1080EIVWDKGRDF1090ATVRKVLSMP1100QVNIVKKTEV1110QTGGFSKESI1120LPKRNSDKLI1130ARKKDWDPKK1140YGGFDSPTVA PCT/US2014/029068 WO 2014/144592 1150 1160 1170 1180 1190 1200YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK1210 1220 1230 1240 1250 1260YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE1270 1280 1290 1300 1310 1320QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA1330 1340 1350 1360PAAFKYFDTT IDRKRYTSTK EVLDATLIHO SITGLYETRI DLSQLGGD (SEQ ID NO :33) In some embodiments, the Cas9 nuclease used herein is at least about 50% identical to the sequence of S. pyogenes Cas9, i.e., at least 50% identical to SEQ ID NO:33. In some embodiments, the nucleotide sequences are about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:33. In some embodiments, any differences from SEQ ID NO:33 are in non-conserved regions, as identified by sequence alignment of sequences set forth in Chylinski et al., RNA Biology 10:5,1-12; 2013 (e.g., in supplementary figure 1 and supplementary table 1 thereof); Esvelt et al., Nat Methods. 2013Nov;10(ll):llI6-21 andFonfara et al.,Nucl. Acids Res. (2014) 42 (4): 2577-2590. [Epub ahead of print 2013 Nov 22] doi:10.1093/nar/gktl074.To determine the percent identity of two sequences, the sequences are aligned for optimal comparison purposes (gaps are introduced in one or both of a first and a second amino acid or nucleic acid sequence as required for optimal alignment, and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 50% (in some embodiments, about 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, or 100% of the length of the reference sequence is aligned). The nucleotides or residues at corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleotide or residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For purposes of the present application, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) WO 2014/144592 PCT/US2014/029068 algorithm which has been incorporated into the GAP program in the GCG software package, using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Cas9-HFD Cas9-HFD are described in a U.S, Provisional Patent Applications USSN 61/799,647, Filed on March 15, 2013, USSN 61/838,148, filed on 6/21/2013, and PCT International Application No. PCT/US14/27335, all of which are incorporated herein by reference in its entirety.The Cas9-HFD are created by fusing a heterologous functional domain (e.g., a transcriptional activation domain, e.g., from VP64 orNF-kB p65), to the N-terminus or C-terminus of a catalytically inactive Cas9 protein (dCas9). In the present case, as noted above, the dCas9 can be from any species but is preferably from S. pyogenes, In some embodiments, the Cas9 contains mutations in the DIO and H840 residues, e.g., D10N/D10A and H840A/H840N/H840Y, to render the nuclease portion of the protein catalytically inactive, e.g., as shown in SEQ ID NO:33 above.The transcriptional activation domains can be fused on the N or C terminus of the Cas9. In addition, although the present description exemplifies transcriptional activation domains, other heterologous functional domains (e.g., transcriptional repressors (e.g., KRAB, ERD, SID, and others, e.g., amino acids 473-530 of the etsrepressor factor (ERF) repressor domain (ERD), amino acids 1-97 of the KRAB domain of KOX1, or amino acids 1-36 of the Mad mSIN3 interaction domain (SID); see Beerli et al., PNAS USA 95:14628-14633 (1998)) or silencers such as Heterochromatin Protein 1 (HP1, also known as swi6), e.g., HPla or HPlp; proteins or peptides that could recruit long non-coding RNAs (IncRNAs) fused to a fixed RNA binding sequence such as those bound by the MS2 coat protein, endoribonuclease Csy4, or the lambda N protein; enzymes that modify the methylation state of DNA (e.g., DNAmethyltransferase (DNMT) or TET proteins); or enzymes that modify histone subunits (e.g., histone acetyltransferases (HAT), histone deacetylases (HDAC), histone methyltransferases (e.g., for methylation of lysine or arginine residues) or histone demethylases (e.g., for demethylation of lysine or arginine residues)) as are known in the art can also be used. A number of sequences for such domains are known in the art, e.g., a domain that catalyzes hydroxylation of methylated cytosines in DNA. Exemplary proteins include the Ten-Eleven- WO 2014/144592 PCT/US2014/029068 Translocation (TET)l-3 family, enzymes that converts 5-methylcytosine (5-mC) to 5- hydroxymethylcytosine (5-hmC) in DNA.Sequences for human TET1-3 are known in the art and are shown in the following table: GenBank Accession Nos. Gene Amino Acid Nucleic Acid TET1 NP 085128.2 NM 030625.2TET2*NP_001120680.1 (var 1)NP 060098.3 (var 2)NM_001127208.2NM 017628.4TET3 NP 659430.1 NM 144993.1 * Variant (1) represents the longer transcript and encodes the longer isoform (a). Variant (2) differs in the 5' UTR and in the 3' UTR and coding sequence compared to variant 1. The resulting isoform (b) is shorter and has a distinct C-terminus compared to isoform a.

In some embodiments, all or part of the full-length sequence of the catalytic domain can be included, e.g., a catalytic module comprising the cysteine-rich extension and the 2OGFeDO domain encoded by 7 highly conserved exons, e.g., the Tetl catalytic domain comprising amino acids 1580-2052, Tet2 comprising amino acids 1290-1905 and Tet3 comprising amino acids 966-1678. See, e.g., Fig. 1 of Iyer et al., Cell Cycle. 2009 Jun l;8(ll):1698-710. Epub 2009 Jun 27, for an alignment illustrating the key catalytic residues in all three Tct proteins, and the supplementary materials thereof (available at ftp site ftp.ncbi.nih.gov/pub/aravind/DONS/supplementary_material_DONS.html ) for full length sequences (see, e.g., seq 2c); in some embodiments, the sequence includes amino acids 1418-2136 of Tetl or the corresponding region in Tet2/3.Other catalytic modules can be from the proteins identified in Iyer et al., 2009.In some embodiments, the heterologous functional domain is a biological tether, and comprises all or part of (e.g., DNAbinding domain from) the MS2 coat protein, endoribonuclease Csy4, or the lambda N protein. These proteins can be used to recruit RNA molecules containing a specific stem-loop structure to a locale specified by the dCas9 gRNA targeting sequences. For example, a dCas9 fused to MS2 coat protein, cndoribonuclcasc Csy4, or lambda N can be used to recruit a long non-coding RNA(lncRNA) such as XIST or HOTAIR; see, e.g., Keryer-Bibens et al., Biol. Cell 100:125-138 (2008), that is linked to the Csy4, MS2 or lambda N binding sequence. Alternatively, the Csy4, MS2 or lambda N protein binding sequence can be linked to another protein, e.g., as described in Keryer-Bibens et al., supra, and the PCT/US2014/029068 WO 2014/144592 protein can be targeted to the dCas9 binding site using the methods and compositions described herein. In some embodiments, the Csy4 is catalytically inactive.In some embodiments, the fusion proteins include a linker between the dCasand the heterologous functional domains. Linkers that can be used in these fusion proteins (or between fusion proteins in a concatenated structure) can include any sequence that does not interfere with the function of the fusion proteins. In preferred embodiments, the linkers are short, e.g., 2-20 amino acids, and are typically flexible (i.c., comprising amino acids with a high degree of freedom such as glycine, alanine, and serine). In some embodiments, the linker comprises one or more units consisting of GGGS (SEQ ID NO:34) or GGGGS (SEQ ID NO:35), e.g., two, three, four, or more repeats of the GGGS (SEQ ID NO:34) or GGGGS (SEQ ID NO:35) unit. Other linker sequences can also be used.

Expression Systems In order to use the guide RNAs described, it may be desirable to express them from a nucleic acid that encodes them. This can be performed in a variety of ways. For example, the nucleic acid encoding the guide RNAcan be cloned into an intermediate vector for transformation into prokaryotic or eukaryotic cells for replication and/or expression. Intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the guide RNA for production of the guide RNA. The nucleic acid encoding the guide RNA can also be cloned into an expression vector, for administration to a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoan cell.To obtain expression, a sequence encoding a guide RNA is typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well !mown in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 2010). Bacterial expression systems for expressing the engineered protein are available in, e.g., E. coll, Bacillus sp., and Salmonella (Paiva et al., 1983, Gene 22:229-235). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.

PCT/US2014/029068 WO 2014/144592 The promoter used to direct expression of a nucleic acid depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of fusion proteins. In contrast, when the guide RNAis to be administered in vivo for gene regulation, either a constitutive or an inducible promoter can be used, depending on the particular use of the guide RNA, In addition, a preferred promoter for administration of the guide RNA can be a weak promoter, such as HSV TK or a promoter having similar activity. The promoter can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Galresponse elements, lac repressor response element, and small molecule control systems such as tetracycline-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547; Oligino et al., 1998, Gene The!5:491-496 ,,־; Wang et al., 1997, Gene Then, 4:432-441; Neering et al., 1996, Blood, 88:1147-55; and Rendahl et al., 1998, Nat. Biotechnol., 16:757-761).In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. Atypical expression cassette thus contains a promoter operably linked, e.g., to the nucleic acid sequence encoding the gRNA, and any signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous spliced intronic signals.The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the gRNA, e.g., expression in plants, animals, bacteria, fungus, protozoa, etc. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and commercially available tag-fusion expression systems such as GST and LacZ.Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SVearly promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

WO 2014/144592 PCT/US2014/029068 The vectors for expressing tine guide RNAs can include RNA Pol III promoters to drive expression of the guide RNAs, e.g., the Hl, U6 or 7SK promoters. These human promoters allow for expression of gRNAs in mammalian cells following plasmid transfection. Alternatively, a T7 promoter may be used, e.g., for in vitro transcription, and the RNA can be transcribed in vitro and purified. Vectors suitable for the expression of short RNAs, e.g., siRNAs, shRNAs, or other small RNAs, can be used.Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. High yield expression systems are also suitable, such as using a baculovirus vector in insect cells, with the gRNA encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.The elements that are typically included in expression vectors also include a replicon that functions in E. coll, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of recombinant sequences.Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., 1989, J. Biol. Chern., 264:17619-22; Guide to Protein Purification, in Methods in Enzymology, vol. 1(Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, 1977, J. Bacterial. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu ct al.,eds, 1983).Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al, supra), It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the gRNA.The present invention includes the vectors and cells comprising the vectors.

PCT/US2014/029068 WO 2014/144592 EXAMPLES The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1. Assessing specificity of RNA-guided endonucleases CRISPR RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. This example describes the use of a human cell-based reporter assay to characterize off-target cleavage of Cas9-based RGNs. Materials and Methods The following materials and methods were used in Example 1. Construction of guide RNAs DNAoligonucleotides (Table A)harboring variable 20 nt sequences for Castargeting were annealed to generate short double-strand DNA fragments with 4 bp overhangs compatible with ligation into BsmBI-digested plasmid pMLM3636. Cloning of these annealed oligonucleotides generates plasmids encoding a chimeric +103 single-chain guide RNA with 20 variable 5’ nucleotides under expression of a U6 promoter (Hwang et al., Nat Biotechnol 31, 227-229 (2013); Mali et al., Science 339, 823-826 (2013)). pMLM3636 and the expression plasmid pJDS246 (encoding a codon optimized version of Cas9) used in this study are both available through the non-profit plasmid distribution service Addgene (addgene.org/crispr-cas ).

Table A qRNA Target Sequence PositionOligos for generating gRN A expression plasmid EGFP Target Site 1 20 19 18 17 16 15 14 13 12 11 10 9 8 ד 6 5 4 3 2 1 oligonucleotide 1 (5' to 3') # oligonucleotide 2 (5' to 3') # G G G 0 A C G G G C A G C T T G C c G G ACACCGGGCACGGGCAGCTTGCCGGG 36. AAAACCCGGCAAGCTGCCCGTGCCCG 230 G G G 0 A C G G G c A G C T T G C c G 6 AC AC CGG GC AC G GG C AGC T T GC C GC G 37 . AAAACGCGGCAAGCIGCCCGTGCCCG 231 G G G 0 A C G G G c A G C T T G C c #6 G ACACCGGGCACGGGCAGCTTGCCGGG 38 . AAAACCCGGCAAGCTGCCCGTGCCCG 232 G G G 0 A 0 G G G c A G C T T G C q G G ACACCGGGCACGGGCAGCTTGCGGGG39. AAAACCCGGCAAGCTGCCCGTGCCCG 233. G G G a A C G G G c A G c T T G c G G ACACCGGGCACGGGCAGCTTGGCGGG 40. AAAACCCGGCAAGCTGCCCGTGCCCG 234 . G G G c A c G G G c A G c T T c c G G ACACCGGGCACGGGCAGCTTCCCGGG 41. AAAACCCGGCAAGCTGCCCGTGCCCG 235. G G G c A 0 G G G c A G c T ؛؛ 8S ®؛ G c c G G ACACCGGGCACGGGCAGCTAGCCGGG 42. AAAACCCGGCTAGCTGCCCGTGCCCG 236. G G G c A c G G G c A G c ؛#؛؛؟؛ T G c c G G ACACCGGGCACGGGCAGCATGCCGGG 43. AAAACCCGGCATGCTGCCCGTGCCCG 237 G G G c A c G G G c A G T T G c c G G ACACCGGGCACGGGCAGGTTGCCGGG44. AAAACCCGGCAAGCTGCCCGTGCCCG 238. G G G c A c G G G c A c T T G c c G G ACACCGGGCACGGGCACCTTGCCGGG 45. AAAACCCGGCAAGGTGCCCGTGCCCG 239 G G G c A c G G G c ؛ w ؛< G 0 T T G c c G G ACACCGGGCACGGGCTGCTTGCCGGG46. AAAACCCGGCAAGCAGCCCGTGCCCG 240. G G G c A a G G G A G c T T G c c G G ACACCGGGCACG3GGAGCTTGCCGGG 47. AAAACCCGGCAAGCTCCCCG’TGCCCG241. G G G c A c G G #6^ c A G c T T G c c G G ACACCGGGCACGGCCAGCTTGCCGGG 48. AAAACCCGGCAAGCTGCCCGTGCCCG 242. G G G c A c G G c A G c T T G c c G G ACACCGGGCACGCGCAGCTTGCCGGG49. AAAACCCGGCAAGCTGCGCGTGCCCG 243 G G G c A c ־־®# G G c A G c T T G c c G G ACACCGGGCACGGGCAGCTTGCCGGG 50. AAAACCCGGCAAGCTGCCGGTGCCCG 244 G G G c A G G G c A G c T T G c c G G ACACCGGGCAGGGGCAGCTTGCCGGG51. AAAACCCGGCAAGCTGCCCGTGCCCG 245. G G G c WS C G G G c A G c T T G c c G G ACACCGGGCTCGGGCAGCTTGCCGGG 52. AAAACCCGGCAAGCTGCCCGAGCCCG 246. G G G A c G G G a A G c T T G c c G G ACACCGGGCACGGGCAGCTTGCCGGG 53. AAAACCCGGCAAGCTGCCCGTGCCCG 247. G G c A a G G G c A G c T T G c c G G ACACCGGGCACGGGCAGCTTGCCGGG 54. AAAACCCGGCAAGCTGCCCGTGCCCG 248 G c G a A c G G G c A G c T T G c c G G ACACCGGGCACGGGCAGCTTGCCGGG 55. AAAACCCGGCAAGCTGCCCGTGCGCG249 G G G c A c G G G c A G c T T G c c ACACCGGGCACGGGCAGCTTGCCCCG 56. AAAACCCGGCAAGCTGCCCGTGCCCG 250. G G G a A 0 G G G c A G c T T G 8:^:8 8p ؛ G G ACACCGGGCACGGGCAGCTTGGGGGG 57. AAAACCCCCCAAGCTGCCCGTGCCCG 251 A G G c A a G G G c A G c T : ؛ w ؟؛ w c c G G ACACCGGGCACGGGCAGCTAGCCGGG 58. AAAACCCGGGTAGCTGCCCGTGCCCG 252 G G G c A c G G G c A G ^؛؛ T G c c G G ACACCGGGCACGGGCAGGATGCCGGG 59. AAAACCCGGCATGCTGCCCGTGCCCG 253 G G G c A c G G G c ■ c T T G c c G G ACACCGGGCACGGGCTGCTTGCCGGG 60. AAAACCCGGCAAGGAGCCCGTGCCCG 254 G G G c A c G G ®cts A G c T T G c c G G ACACCGGGCACGGCGAGCTTGCCGGG 61. AAAACCCGGCAAGCTCGCCGTGCCCG 255 G G G c A c # c ®؛ 8 G c A G c T T G c c G G ACACCGGGCACGCGCAGCTTGCCGGG 62. AAAACCCGGCAAGCTGCGCGTGCCCG 256 G G G c Wit G G G c A G c T T G c c G G ACACCGGGCTGGGGCAGCTTGCCGGG63. AAAACCCGGCAAGCTGCCCGAGCCCG 257. G G w ؛ 8 ؛ ®Sep A C G G G c A G c T T G c c G G ACACCGGCGACGGGCAGCTTGCCGGG64. AAAACCCGGCAAGCTGCCCGTCGCCG 258 G ؛ W88 c A C G G G 0 A G c T T G c c G G ACACCGCCCACGGGCAGCTTGCCGGG 65. AAAACCCGGCAAGCTGCCCGTGCGCG 259. G ؛ Wf8 A c G G G c A G c T T G c c G G ACACCGGCGACGGGCAGCTTGCCGGG 66. AAAACCCGGCTAGCTGCCCGTGCCCG 260 . G N# c G G G c A G c T T G c c G G ACACCGCCGTCGGGCAGCTTGCCGGG 67. AAAACCCGGCAAGCTGCCCGTGCCCG 261 G c : ؛؛ w ؛؛ 9 # WS G G G c A G c T T G c c G G ACACCGCCGTGGGGCAGCTTGCCGGG 68. AAAACCCGGCAAGCTGCCCGTGCCCG 262. G <®i8s ■sig® G G c A G c T T G c c G G ACACCGCCGTGCGGCAGCTTGCCGGG 69. AAAACCCGGCAAGCTGCCCGTGCCCG 263. G ؛؟؟،؛# N W G c A G c T T G c c G G ACACCGCCGTGCCGCAGCTTGCCGGG70. AAAACCCGGCAAGCTGCCCGTGCCCG 264. G ؛؛; w w ؛؛< iS8ei® c A G c T T G c c G G ACACCGCCGTGCCCCAGCTTGCCGGG71. AAAACCCGGCAAGCTGCCCGTGCCCG 265 G 8888g? %S8$ #6# ؛؛؛»#؛ x®C# ؛ p ;# A G c T T G c c G G ACACCGCCGTGCCCGAGCTTGCCGGG72. AAAACCCGGCAAGCTGCCCGTGCCCG 266 G G G c A 0 G G G c A G c T T G c G 88W ACACCGGGCACGGGCAGCTTGCGGGG 73 . AAAACGCCGCAAGCTGCCCGTGCCCG 267 G G G c A c G G G c A G c T T ■ c !8988 G G ACACCGGGCACGGGCAGCTTGCGGGG 74. AAAACCCCGGAAGCTGCCCGTGCCCG 268 W O 2014/144592 ، n PCT/US2014/029068 Table A qRNA Target Sequence Position Oliqos for generating gRN A expression plasmid G G G C A c G G G C A G C T G c G G ACACCGGGCACGGGCA.GCATGCGGGG 75 . AAAACCCCGCATGCTGCCCGTGCCCG 269 G G G C A c G G G C A C T T G c ף G G ACACCGGGCACGGGCACCTTGCGGGG 76. AAAACCCCGCAAGGTGCCCGTGCCCG 270 G G G c A c G G G ®ft® A G c T T G c fl G G ACACCGGGCACGGGGAGCTTGCGGGG 77. AAAACCCCGCAAGCTCCCCGTGCCCG 271. G G G c A c G ®ft® G C A G c T T G c fl G G ACACCGGGCACGCGCAGCTTGCGGGG 78. AAAACCCCGCAAGCTGCGCGTGCCCG 272 G G G c A ®:ft® G G G c A G c T T G c G G ACACCGGGCAGGGGCAGCTTGCGGGG 79. AAAACCCCGCAAGCTGCCCCTGCCCG 273 G G G A c G G G c A G c T T G c q G G ACACCGGGGACGGGCAGCTTGCGGGG 80. AAAACCCCGCAAGCTGCCCGTCCCCG 274 G G C A c G G G c A G a T T G c G G ACACCGCGCACGGGCAGCTTGCGGGG 81. AAAACCCCGCAAGCTGCCCGTGCGCG 275 G G G c A c G G G N® A G c T T G c c G NN ACACCGGGCACGGGGAGCTTGCCGCG 82. AAAACGCGGCAAGGTCCCCGTGCCCG 276 G G G c A c G G G ®ft® A G c T T c c G G ACACCGGGCACGGGGAGCTTCCCGGG 83. AAAACCCGGGAAGCTCCCCGTGCCCG277 G G G c A c G G G ®ft® A G c T G c c G G ACACCGGGCACGGGGAGCATGCCGGG 84 . AAAACCCGGCATGCTCCCCGTGCCCG 278 G G G c A c G G G A ؛؛ w c T T G c c G G ACACCGGGCACGGGGACCTTGCCGGG 85. AAAACGCGGCAAGGTCCCCGTGCCCG 279. G G G c A c G G ®ft® A G c T T G c c G G ACACCGGGCACGCGGAGCTTGCCGGG 86. AAAACCCGGCAAGCTCCGCGTGCCCG 280 G G G c A G G G A G c T T G c c G G ACACCGGGCAG GGGG AGC T T GCC GGG 87 . AAAACCCGGCAAGCTCCCCCTGCCCG 281.®::ft:® G G G ®:ft® A c G G G ®ft® A G c T T G c c G G ACACCGGGGACGGGGAGCTTGCCGGG 88. AAAACCCGGCAAGCTCCCCGTCCCCG 282. G ’ O ؛؛® G c A c G G G A G c T T G c c G G ACACCGCGCACGGGGAGCTTGCCGGG 89. AAAACCCGGCAAGCTCCCCGTGCGCG 283. G c G c A c G G G c A G c T T G c c G ACACCGCGCACGGGCAGCTTGCCGCG 90. AAAACGCGGCAAGCTGCCCGTGCGCG 284. G c G c A c G G G c A G c T T c c G G ACACCGCGCACGGGCAGCTTCCCGGG 91. AAAACCCGGGAAGCTGCCCGTGCGCG 285 . G c G c A c G G G c A G c T G c c G G ACACCGCGCACGGGCAGCATGCCGGG 92. AAAACCCGGCATGCTGCCCGTGCGCG 286. G G c A c G G G c A c c T T G c c G G ACACCGCGCACGGGCACCTTGCCGGG 93. AAAACCCGGCAAGGTGCCCGTGCGCG 287 G G c A c G ®ft® G c A G c T T G c c G G ACACCGCGCACGCGCAGCTTGCCGGG 94. AAAACCCGGCAAGCTGCGCGTGCGCG 289. G G c A ®ft® G G G c A G c T T G c c G G ACACCGCGCAGGGGCAGCTTGCCGGG 95 . AAAACCCGGGAAGCTGCCCGTGCGCG 289. G G ®ft® A c G G G c A G c T T G c c G G ACACCGCGGACGGGCAGGTTGCCGGG 96. AAAACGCGGCAAGCTGCCCGTGCGCG 290 EGFP Target Site 2 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 3')oligonucleotide 2 (5' to 3') G A T G C c G T T c T T c T G C T T G T ACACCGATGCCGTTCTTCTGCTTGTG 97. AAAACACAAGCAGAAGAACGGCATCG 291 G A T G C c G T T c T T c T G C T T G ACACCGATGCCGTTCTTCTGCTTGAG 98. AAAACACAAGCAGAAGAACGGCATCG 292 G A T G c c G T T c T T c T G C T T W T ACACCGATGCCGTTCTTCTGCTTCTG 99. AAAACACAAGCAGAAGAACGGCATCG 293 G A T G c c G T T c T T c T G C T ma G T ACACCGATGCCGTTCTTCTGCTAGTG100 AAAACACZ1AGCAGAAGAACGGCATCG 294 G A T G c c G T T c T T c T G C SO T G T ACPCCGATGCCGTTCTTCTGCATGTG101 AAAACACAAGCAGAAGAACGGCATCG 295 G A T G c c G T T c T T c T G ::ifts: T T G T ACACCGATGCCGTTCTTCTGGTTGTG 102 AAAACACAAGCAGAAGAACGGCATCG 296. G A T G c c G T T c T T c T Ne C T T G T ACACCGATGCCGTTCTTCTCCTTGTG 103 AAAACACAAGCAGAAGAACGGCATCG 297 . G A T G c c G T T c T T c G c T T G T ACACCGATGCCGTTCTTCAGCTTGTG 104 AAAACAC AAGC AG71AGAACGGC AT C G 298. G A T G c c G T T c T T T G c T T G T ACACCGATGCCGTTCTTGTGCTTGTG105 AAAACACAAGCAGAAGAACGGCATCG 299. G A T G c c G T T c T ؛ W ؛؛ c T G c T T G T ACACCGATGCCGTTCTACTGCTTGTG106 AAAACACAAGCAGAAGAACGGCATCG 300. G A T G c c G T T c ;Sia® T c T G c T T G T ACACCGATGCCGTTCATCTGCTTGTG 107 AAAACACAAGCAGAA.GAACGGCATCG 301. G A T G c c G T T T T c T G c T T G T ACACCGATGCCGTTGTTCTGCTTGTG108 AAAACACAAGCAGAAGAACGGCATCG 302. :::::ft::::: G A T G c c G T ?®a® c T T c T G c T T G T ACACCGATGCCGTACTTCTGCTTGTG 109 AAAACACAAGCAGAAGAACGGCATCG 303. G A T G c c G O T c T T c T G c T T G T ACACCGATGCCGA’TCTTCTGCTTGTG110 AAAACACAAGCAGAAGAACGGCATCG 304. G A T G c c T T c T T c T G c T T G T ACACCGATGCCCTTCTTCTGCTTGTG 111 AAA71.CACAAGCAGAAGAA-CGGCATCG 305 G A T G c ®fl® G T T c T T c T G c T T G T ACACCGATGCGGTTCTTCTGCTTGTG 112 AAAACACAAGCAGAAGAACGGCATCG 306 G A T G IW c G T T c T T c T G c T T G T ACACCGATGGCGTTCTTCTGCTTGTG 113 AAAACACAAGCAGAAGAACGGCATCG 307.

W O 2014/144592 .< PCT/US2014/029068 Table A qRNA Target Sequence Position Oliqos for qeneratinq qRNA expression plasmid G A T C c G T T C T T C T G C T T G T ACACCGATCCCGTTCTTCTGCTTGTG 114 AAAACACAAGCAGAAGAACGGCATCG 308. G A G C c G T T c T T C T G C T T G T ACACCGAAGCCGTTCTTCTGCTTGTG 115 AAAACACAAGCAGAAGAACGGCATCG 309. G T G C c G T T c T T c T G C T T G T ACACCGTTGCCGTTCTTCTGCTTGTG 116 AAAACACAAGCAGAAGAACGGCATCG 310 . G A T G C c G T T c T T c T G C T T e SO ACACCGATGCCGTTCTTCTGCTTCAG 117 AAAACTGAAGCAGAAGAACGGCATCG . ־ 31 G A T G C c G T T c T T c T G C SO G T ACACCGATGCCGTTCTTCTGCAAGTG 118 AAAACACAAGCAGAAGAACGGCATCG 312. G A T G C c G T T c T T c T ؛ W •؛ T T G T ACACCGATGCCGTTCTTCTCGTTGTG 119 AAAACACAAGCAGAAGAACGGCATCG 313 G A T G C c G T T c T T p ؛ s G c T T G T ACACCGATGCCGTTCTTGAGCTTGTG 120 AAAACACAAGCTCAAGAACGGCATCG 314 G A T G C c G T T c so c T G c T T G T ACACCGATGCCGTTCAACTGCTTGTG 121 AAAACACAAGCAGAAGAACGGCATCG 315. G A T G c c G T S ؛ Sa w T T c T G c T T G T ACACCGATGCCGTAGTTCTGCTTGTG 122 AAAACACAAGCAGAAGAACGGCATCG 316. G A T G c c kO ؛؟s$|?s T c T T c T G c T T G T ACACCGATGCCCATCTTCTGCTTGTG 123 AAAACACAAGCAGAAGAACGGCATCG 317. G A T G ״J- G T T c T T c T G c T T G T ACACCGATGGGGTTCTTCTGCTTGTG 124 AAAACACAAGCAGAAGAACGGCATCG 318. G A ^ S ؛ S^S c c G T T c T T c T G c T T G T ACACCGAACCCGTTCTTCTGCTTGTG 125 AAAACACAAGCAGAAGAA.CGGCATCG 319. G ■ S ؟ g ؛ s G c c G T T c T T c T G c T T G T ACACCGTAGCCGTTCTTCTGCTTGTG 126 AAAACACAAGCAGAAGAACGGCAAGG 320. G SSO SO c c G T T c T T c T G c T T G T ACACCGTACCCGTTCTTCTGCTTGTG 127 AAAACACAAGCAGAAGAACGGGTACG 321. G t - ■ c G T T c T T c T G c T T G T ACACCGTACGCGTTCTTCTGCTTGTG 128 AAAACACAAGCAGAAGAACGCGTACG 322. G SSasS: SfF ■ G T T c T T c T G c T T G T ACACCGTACGGGTTCTTCTGCTTGTG 129 AAAACACAAGCAGAAGAACCCGIACG 323. G W; SiStS T T c T T c T G c T T G T ACACCGTACGGCTTCTTCTGCTTGTG 130 AAAACACAAGCAGAAGAAGCCGTACG 324. G ?w SSSs SscS <1 T c T T c T G c T T G T ACACCGTACGGCATCTTCTGCTTGTG 131 AAAACACAAGCAGAAGATGCCGTACG 325 G SSSsS fl ؛؛ w ؛؛ Sei ؛; gS ؛ S ؛ a c T T c T G c T T G T ACACCGTACGGCAACTTCTGCTTGTG 132 AAAACACAAGCAGAAGTTGCCGTACG 326 G S58S; SiBS RssSieS ?Sass w* T T c T G c T T G T ACACCGTACGGCAAGTTCTGCTTGTG 133 AAAACACAAGCAGAACTTGCCGTACG 327. G A T G c c G T T c T T c T G c T sW G ؟ w ؛؛ ACACCGPTGCCGTTCTTCTGCTAGAG 134 AAAACTCTAGCAGAAGAACGGCATCG 328 G A T G c c G T T c T T c T G V T SO G T ACACCGATGCCGTTCTTCTGGTAGTG 135 AAAACACTACCAGAAGAACGGCATCG 329 G A T G c c G T T c T T c SO; G c T a G T ACACCGATGCCGTTCTTCAGCTAGTG 136 AAAACACTAGCTGAAGAACGGCATCG 330. G A T G c c G T T c T W c T G c T SO G T ACACCGATGCCGTTCTACTGCTAGTG 137 AAAACACTAGCAGTAGAACGGCATCG 331. G A T G c c G T T T T c T G c I SO G T ACACCGATGCCGTTGTTCTGCEAGTG 138 AAAACACTAGCAGAACAACGGCATCG 332. G A T G c c G T c T T c T G c T S8S G T ACACCGATGCCGATCTTCTGCTAGTG 139 AAAACA.CTAGCAGAAGATCGGCATCG 333. G A T G c G T T c T T c T G c T ״as G T ACACCGATGCGGTTCTTCTGCTAGTG 140 AAAACACTAGCAGAAGAACCGCATCG 334. G A T c c c G T T c T T c T G c T ؛؛؟ a ؛ s G T ACACCGATCCCGTTCTTCTGCTAGTG 141 AAAACACTAGCAGAACAACGGCATCG 335. G :، T G c c G T T c T T c T G c T 50 G T ACACCGTTGCCGTTCTTCTGCTAGTG 142 AAAACACTAGCAGAAGAACGGCAACG 336. G A T G c c G T T q T T c T G c T T G ACACCGATGCCGTTGTTCTGCTTGAG 143 AAAACTCAAGCAGAACAACGGCATCG 337 . G A T G c c G T T T T c T G T T G T ACACCGATGCCGTTGTTCTGGTTGTG 144 AAAACACAACCAGAACAACGGCATCG 338 . G A T G c c G T T q T T c G c T T G T ACACCGATGCCGTTGTTCAGCTTGTG 145 AAAACACAAGCTGAACAACGGCATCG 339 G A T G c c G T T A® T O ؟ c T G c T T G T ACACCGATGCCGTTGTACTGCTTGTG 146 AAAACACAAGCAGTACAACGGCATCG 340 G A T G c c G T T T c T G c T T G T ACACCGATGCCGATGTTCTGCTTGTG 147 AAAACACAAGCAGAACATCGGCATCG 341 G A T G c ؟ G T T T T c T G c T T G T ACACCGATGCGGTTGTTCTGCTTGTG 148 AAAACACAAGCAGAAGAACGGCATCG 342 G A T c c G T T T T c T G c T T G T ACACCGATCCCGTTGTTCTGCTTGTG 149 AAAACACAAGCAGAAGAACGGCATCG 343. G T G c c G T T q T T c T G c T T G T ACACCGTTGCCGTTGTTCTGCTTGTG 150 AAAACACAAGCAGAAGAACGGCAAGG 344. G :As T G c c G T T c T T c T G c T T G :::::a::::: ACACCGTTGCCGTTCTTCTGCTTGAG 151 TkAAAC T C AAGC AGAAGAACGGC AACG 345. G ■ T G c c G T T c T T c T G Sp T T G T ACACCGTTGCCGTTCTTCTGGTTGTG 152 AAAACACAACCAGAAGAACGGCAACG 346 G ؛، T G c c G T T c T T c G c T T G T ACACCGTTGCCGTTCTTCAGCTTGTG 153 AAAACACAAGCTGAAGA?.CGGCAACG 347 G T G c c G T T c T « c T G c T T G T ACACCGTTGCCGTTCTACTGCTTGTG 154 AAAACACAAGCAGTAGAACGGCAACG 348 G ،؛ T G a c G T c T T c T G c T T G T ACACCGTTGCCGATCTTCTGCTTGTG 155 AAAACACAAGCAGAAGATCGGCAACG 349. G t T G c Sip G T T c T T c T G c T T G T ACACCGTTGCGGTTCTTCTGCTTGTG 156 AAAACACAAGCAGAAGAACCGCAACG 350.

W O 2014/144592 PCT/US2014/029068 Table A qRNA Target Sequence Position Oliqos for generating gRN A expression plasmid R T w® C C G T T C T T C T G C T T G T ACACCGTTCCCGTTCTTCTGCTTGTG 157 AAAACACAAGCAGAAGAACGGGAACG 351EGFP Target Site 3____________________________________________________________________________________________________________?0 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 3')oligonucleotide 2 (5' to 3')R R T G G T G C A G A T G A A C T T C A ACACCGGTGGTGCAGATGAACTTCAG158 AAAACTGAAGTTCATCTGCACCACCG 352.R G T G G T G C A G A T G A A C T T C « ACACCGGTGGTGCAGATGAACTTCTG 159 AAAACAGAAGTTCATCTGCACCACCG 353. R G T G G T G C A G A T G A A C T T A ACACCGGTGGTGCAGATGAACTTGAG 160 AAAACTGAAGTTCATCTGCACCACCG 354.R R T R G T G C A G A T G A A C T 08 c A ACACCGGTGGTGCAGATGAACTACAG161 AAAACTGTAGTTCATCTGCACCACCG 355 R G T G G T G C A G A T G A A C 08 T c A ACACCGGTGGIGCAGATGAACATCAG 162 AAAACTGATGTTCATCTGCACCACCG 356.R R T R R T G 0 A G A T G A A 08 T T c A ACACCGGTGGTGCAGATGAAGTTCAG163 AAAACTGAACTTCATCTGCACCACCG357. R R T R R T G C A G A T G A 05 c T T c A ACACCGGTGGTGCAGATGATCTTCAG 164 AAAACTGAAGATCATCTGCACCACCG 358. R R T R G T G C A G A T G ؛؛؛؛؛؟؛ A c T T c A ACACCGGTGGTGCAGATGTACTTCAG165 AAAACTGAAGTACATCTGCACCACCG359.R R T G G T G C A G A T c A A c T T c A ACACCGGTGGTGCAGATGAACTTCAG 166 AAAACTGAAGTTGA.TCTGCACCACCG 360R R T G G T G C A G A d G A A c T T c A ACACCGGTGGTGCAGAAGAACTTCAG 167 AAAACTGAAGTTCTTCTGCACCACCG 361R R T G G T G C A G t T G A A c T T c A ACACCGGTGGTGCAGTTGAACTTCAG168 AAAACTGAAG’TICAACTGCACCACCG 362R R T G G T G C A A T G A A c T T c A ACACCGGTGGTGCAGATGAACTTCAG 169 AAAACTGAAGTTCATGTGCACCACCG 363 R R T R G T G c ؛؟؛ w G A T G A A c T T c A ACACCGGTGGTGCTGATGAACTTCAG170 AAAACTGAAGTTCATCAGCACCACCG 364.R G T G G T G A G A T G A A c T T c A ACACCGGTGGTGGAGATGAACTTCAG171 AAAACTGAAGTTCATCTCCACCACCG 3 65.R R T G G T c A G A T G A A c T T c A ACACCGGTGGTCCAGATGAACTTCAG 172 AAAACTGAAGTTCATCTGGACCACCG 3 6 6.R G T G G G c A G A T G A A c T T c A ACACCGGTGGAGCAGATGAACTTCAG173 AAAACTGAAGTTCATCTGCTCCACCG367.R G T G o® T G c A G A T G A A c T T c A ACACCGGTGCTGCAGATGAACTTCAG174 AAAACTGAAGTTCATCTGCAGCACCG 368 R R T C G T G c A G A T G A A c T T c A ACACCGGTCGTGCAGATGAACTTCAG175 AAAACTGAAGTTCATCTGCACGACCG 369R R R R T G c A G A T G A A c T T c A ACACCGGAGGTGCAGATGAACTTCAG 17 6 AAAACTGAAGTTCATCTGCACCTCCG 370R T R R T G c A G A T G A A c T T c A ACACCGCTGGTGCAGATGAACTTCAG177 AAAACTGAAGTTCATCTGCACCAGCG 371R G T G G T G c A G A T G A A c T T 5 ؛ 55q : 55 ^ 5 ؛ ACACCGGTGGTGCAGATGAACTTGTG178 AAAACAGAAGTTCATCTGCACCACCG 372R R T R G T G c A G A T G A A c 55538; 05: c A ACACCGG T GGTGCAGATGAACAACAG 179 AAAACTGTTGTTCATCTGCACCACCG 373R R T R G T G 0 A G A T G A ؛ S5 ؛؛؛ T T c A ACACCGGTGGTGCAGATGATGTTCAG180 AAAACTGAAGATCATCTGCACCACCG 374.R R T G G T G c A G A T tts A c T T c A ACACCGGTGGTGCAGATCTACTTCAG 181 AAAACTGAAGTACATCTGCACCACCG 375.R R T R G T G c A G 05 G A A c T T c A ACACCGGTGGTGCAGTAGAACTTCAG 182 AAAACTGAAGTTCTACTGCACCACCG 376R G T G G T G c ■ A T G A A c T T c A ACACCGGTGGTGCTGATGAACTTCAG 183 AAAACTGAAGTTCATGAGCACCACCG 377 .R R T G G T A G A T G A A c T T c A ACACCGGTGGTGGAGATGAACTTCAG 184 AAAACTGAAGTTCATCTCGACCACCG 378.R R T R ؛® o sss$ G c A G A T G A A c T T c A ACACCGGTGGAGCAGATGAACTTCAG 185 AAAACTGAAGTTCATCTGCTGCACCG 379 R R ؛® se ؛ G T G a A G A T G A A c T T c A ACACCGGAGGTGCAGATGAACTTCAG 186 AAAACTGAAGTTCATCTGCACGTCCG 380R :555^® ؟؛؛ a ؛^ G G T G c A G A T G A A c T T c A ACACCGGAGGTGCAGATGAACTTCAG 187 AAAACTGAAGTTCATCTGCACCAGGG 381. G G T G c A G A T G A A c T T c A ACACCGGAGGTGCAGATGAACTTCAG 188 AAAACTGAAGTTCATCTGCACGTGCG382R T G c A G A T G A A c T T c A ACACCGCACCTGCAGATGAACTTCAG 189 AAAACTGAAGTTCATCTGCAGGTGCG 383.R o Sias? ^^5 5$5e& G c A G A T G A A c T T c A ACACCGCACCAGCAGATGAACTTCAG 190 AAAACTGAAGTTCATCTGCTGGTGCG 384R 8^w* ؛؛؛ o c A G A T G A A c T T c A ACACCGCACCACCAGATGAACTTCAG 191 AAAACTGAAGTTCATCTGGTGGTGCG385RO® i®S2§ SSqsi: A G A T G A A c T T c A ACACCGCACCACGAGATGAACTTCAG 192 AAAACTGAAGTTCATCTCGTGGTGCG386. R W® ®ס־:site w; G A T G A A c T T c A ACACCGCACCACGTGATGAACTTCAG 193 AAAACTGAAGTTCATCACGTGGTGCG 387.R ws ؛ W ؛ 5 ؛ ؛ W ؛؛ iiy 85968? A T G A A c T T c A ACACCGCACCACGTCATGAACTTCAG 194 AAAACTGAAGTTCATGACGTGGTGCG 388 . G G T G G T G c A G A T G A A c T 80 c t ACACCGGTGGTGCAGATGAACTACTG 195 AAAACAGTAGTTCATCTGCACCACCG 389.

W O 2014/144592 _ PCT/US2014/029068 Table A qRNA Tarqet Sequence Position Oligosfor generating gRN A expression plasmid G G T G G T G C A G A T G A A T sW C A ACACCGGTGGTGCAGATGAAGTACAG 196 AAAACTGTACTTCATCTGCACCACCG 390 G G T G G T G C A G A T G w A C T saii C A ACACCGGTGGTGCAGATGTACTACAG197 AAAACTGTAGTACATCTGCACCACCG 391. G G T G G T G C A G A G A A C T C A ACACCGGTGGTGCAGAAGAACTACAG 198 AAAACTGTAGTTCTTCTGCACCACCG 392. G G T G G T G C A A T G A A c T C A ACACCGGTGGTGCACATGAACTACAG199 AAAACTGTAGTTCATGTGCACCACCG 393. G G T G G T a G A G A T G A A c T W5 C A ACACCGGTGGTGGAGATGAACTACAG200 AAAACTGTAGTTCATCTCCACCACCG 3 94 G G T G G G C A G A T G A A c T a C A ACACCGGTGGAGCAGATGAACTACAG 201 AAAACTGTAGIIITCATCTGCTCCACCG 395. G G T G T G C A G A T G A A c T C A ACACCGGTCGTGCAGATGAACTACAG202 AAAACTGTAGTTCATCTGCACGACCG 396 G T G G T G C A G A T G A A c T C A CA.CCGCTGGTGCAGATGAACTACAG 203 AAAACTGTAGTTCATCTGCACCAGCG397 G G T G G T G C A A T G A A c T T c £ A.CCGGTGGTGCACATGAACTTCTG 204 AAAACAGAAGTTCATGTGCACCACCG 398 G G T G G T G 0 A *W A T G A A s:،fs T T c A CACCGGTGGTGCACATGAAGTTCAG 205 AAAACTGAACTTCATGTGCACCACCG 399. G G T G G T G C A A T G w A c T T c A ACACCGGTGGTGCACATGTACTTCAG 206 AAAACTGAAGTACATGTGCACCACCG 400. G G T G G T G C A A W* G A A c T T c A ACACCGGTGG T GCAC AAG AACT T C AG 207 AAAACTGAAGTTCTTGTGCACCACCG 401. G G T G G T G A A T G A A c T T c A ACACCGGTGGTGGACATGAACTTCAG 208 AAAACTGAAGTTCATGTCCACCACCG 402. G G T G G ;siSss G C A ،* A T G A A c T T c A ACACCGGTGGAGCACATGAACTTCAG209 AAAACTGAAGTTCATGTGCTCCACCG 403 G G T G T G C A e A T G A A c T T c A ACACCGGTCGTGCACATGAACTTCAG 210 AAAACTGAAGTTCATGTGCACGACCG 404. G T G G T G c A c A T G A A c T T c A ACCGCTGGTGCACATGAACTTCAG211 AAAACTGAAGTTCATGTGCACCAGCG 405 G 168s־ T G G T G c A G A T G A A c T T c i JACCGCTGGTGCAGATGAACTTCTG212 AAAACA.GAAGTTCATCTGCACCAGCG406. G T G G T G c A G A T G A A 8^8 T T c A ACACCGCTGGTGCAGATGAAGTTCAG213 AAAACTGAACTTCATCTGCACCAGCG 407. G se T G G T G c A G A T G A c T T c A ACA.CCGCTGGTGCAGATGTACTTCAG214 AAAACTGAAGTACATCTGCACCAGCG 408 G Ws T G G T G c A G A ؛؛ w G A A c T T c A ACACCGCTGGTGCAGAAGAACTTCAG 215 AAAACTGAAGTTCTTCTGCACCAGCG 409 G T G G T G A G A T G A A c T T c A ACACCGCTGGTGGAGATGAACTTCAG 216 AAAACTGAAGTTCATCTCCACCAGCG 410 G T G G 8^8 G c A G A T G A A c T T c A ACACCGCTGGAGCAGATGAACTTCAG 217 AAAACTGAAGTTCATCTGCTCCAGCG 411 G T G T G c A G A T G A A c T T c A ACACCGCT CGTGCAGATGAACT T CAG 218 AAAACTGAAGTTCATCTGCACGAGCG 412 . Endoqenous Tarqet 1 (VEGFA Site 1) ?0 10 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5’ to 3’)oligonucleotide 2 (5' to 3') G G G T G G G G G G A G T T T G C T c C ACACCGGGTGGGGGGAGTTTGCTCCG219 AAAACGGAGCAAACTCCCCCCACCCG 413.220 414 Endoqenous Tarqet 2 (VEGFA Site 2): 20 19 18 17 16 15 14 13 12 11 10 9 8 ד 6 5 4 3 2 1 oligonucleotide 1 (5' to 3')oligonucleotide 2 (5' to 3’) G A a a C C C T C C A C C c C G C C T C ACACCGACCCCCTCCACCCCGCCTCG 221 AAAACGAGGCGGGGTGGAGGGGGTCG415222 416. Endoqenous Tarqet 3 (VEGFA Site 3): _________________ __ ________ _______________________________________________ _____ 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 3')oligonucleotide 2 (5' to 3') G G T G A G T G A G T G T G T G C G T G ACACCGGTGAGTGAGTGTGTGCGTGG 223 AAAACCACGCACACACTCACTCACCG417.224 418 Endoqenous Tarqet 4 (EMX1Y. 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 3')oiigonucieotiae z <כ־ to 3') G A G T C C G A G 0 A G A A G A A G A A ACACCGAGTCCGAGCAGAAGAAGAAG 225 AAAACTTCCTCTTCTGCTCGGACTCG 419 W O 2014/144592 , , PCT/US2014/029068 Table A gRNA Target Sequence Position Oliqos for generating gRNA expression plasmid i • I 226]420. Endogenous Target 5 (RNF2): 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 3')oligonucleotide 2 (5'־( 3to G T C A T C T T A G T C A T T A C C T G ACACCGTCATCTTAGTCATTACCTGG 227 AAAACCAGGTAATGACTAAGATGACG 421.228 422 Endogenous Target 6 (FAWCF): 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 3')oligonucleotide 2 (5' 3')to G G A A T C C C T T C T G C A G C A C C ACACCGGAATCCCTTCTGCAGCACCG 229 AAAACGGTGCTGCAGAAGGGATTCCG 423Sequences of 0 igonucleotides used to generate expression plasmids encoding single gRNAs/variant single gRNAs targeted to sites inthe EGFP reporter gene and single gRNAs targeted to six endogenous human gene targets. #, SEQ ID NO:.

W O 2014/144592 Ac PCT/US2014/029068 PCT/US2014/029O68 WO 2014/144592 EGFP Activity Assays U20S.EGFP cells harboring a single integrated copy of an EGFP-PEST fusion gene were cultured as previously described (Reyon et al., Nat Biotech 30, 460- 465 (2012)). For transfections, 200,000 cells were Nucleofected with the indicated amounts of sgRNA expression plasmid and pJDS246 together with 30 ng of a Td- tomato-encoding plasmid using the SE Cell Line 4D-Nucleofector™ X Kit (Lonza) according to the manufacturer ’s protocol. Cells were analyzed 2 days post- transfection using a BD LSRII flow cytometer. Transfections for optimizing gRNA/Cas9 plasmid concentration were performed in triplicate and all other transfections were performed in duplicate, PCR amplification and sequence verification of endogenous human genomic sites PCR reactions were performed using Phusion Hot Start II high-fidelity DNA polymerase (NEB) witli PCR primers and conditions listed in Table B.Most loci amplified successfully using touchdown PCR (98 °C, 10 s; 72-62 °C, -1 °C/cycle, s; 72 °C, 30 s] 10 cycles, [98 °C, 10 s; 62 °C, 15 s; 72 °C, 30 s]25 cycles). PCR for the remaining targets were performed with 35 cycles at a constant annealing temperature of 68 °C or 72 °C and 3% DMSO or IM betaine, if necessary. PCR products were analyzed on a QIAXCEL capillary electrophoresis system to verify both size and purity. Validated products were treated witli ExoSap-IT (Affymetrix) and sequenced by the Sanger method (MGH DNA Sequencing Core) to verify each target site.

TABLE B Actual Target in U20S.EGFP cells Forward PCR Primer SEQID NO Reverse PCR Primer SEQ IDNO:PCR ConditionsWatson- Crick Trans- versions non- Watson -Crick Trans- version s Tran- sitions TCCAGATGGCACATTGTCAG 436. AGGGAGCAGGAAAGTGAGGT 748. DMSOGGGGCCCACTCTTCTTCCAT 437. ACCCAGACTCCTGGTGTGGC 749. No DMSO 0 0 1GCTAAGCAGAGATGCCTATGCC 438. ACCACCCTTTCCCCCAGAAA 750. DMSO 2 0 0؛،،،،؛ii■،،،، ،»،GCATGTCAGGATCTGACCCC 441. TGCAGGGCCATCTTGTGTGT 753. DMSO 0 2 01GGCTCTCCCTGCCCTAGTTT 443 . GCAGGTCAAGTTGGAACCCG 755. DMSO 0 2 1GGGGCTGAGAACACATGAGATGCA 444 . AGATTTGTGCACTGCCTGCCT 756. DMSO 1 0 2CCCGACCTCCGCTCCAAAGC 445. GGACCTCTGCACACCCTGGC 757. DMSO 2 1 0TGCAAGGTCGCATAGTCCCA 446. CAGGAGGGGGAAGTGTGTCC 758. DMSO 1 1 1755*■BillGCCCCCAGCCCCTCTGTTTC 448. GCTGCTGGTAGGGGAGCTGG 760. DMSO 1 2 0CGGCTGCCTTCCCTGAGTCC 449. GGGTGACGCTTGCCATGAGC 761.72C Anneal, 3% DMSO2 0TGACCCTGGAGTACAAAATGTTCCCA 450 . GCTGAGACAACCAGCCCAGCT 762.72C Anneal, 3% DMSO1 0TGCCTCCACCCTTAGCCCCT 451. GCAGCCGATCCACACTGGGG 763. DMSO 1 0 2AACT CAGGACAACACT GCC T GT 452. CCCAGGAGCAGGG TAGAATGC 764. DMSO 0 1 2TCCTCCTTGGAGAGGGGCCC 453. CCTTGGAAGGGGCCTTGGTGG 765. DMSO 0 3 0CCGAGGGCATGGGCAATCCT 454. GGCTGCTGCGAGTTGCCAAC 766. DMSO 0 1 3TGCTTTGCATGGGGTCTCAGACA 455. GGGTTGCTTGCCCTCTGTGT 767. DMSO 0 2 2AGCTCCTTCTCATTTCTCTTCTGCTGT 456. CACAGAAGGATGTGTGCAGGTT 768. DMSO 0 2 2AGCAGACACA.GGTGAATGCTGCT 457. GGTCAGGTGTGCTGCTAGGCA 769. DMSO 1 1 2CCTGTGGGGCTCTCAGGTGC 458 . ACTGCCTGCCAAAGTGGGTGT 770. No DM5OTD 1 1 2AGCTGCACTGGGGAATGAGT 459 . TGCCGGGTAATAGCTGGCTT 771. DMSO 0 1 3CCAGCCTGGGCAACAAAGCG 460. GGGGGCTTCCAGGTCACAGG 772.72C Anneal, 3% DMSO, 6% DMSO3 1 TACCCCCACTGCCCCATTGC 461.ACAGGTCCATGCTTAGCAGAGG G773. DMSO 0 1 3 W O 2014/144592 PCT/US2014/029068 TABLE B Actual Target in U2OS.EGFP cells Forward PCR PrimerSEQ ID NO Reverse PCR Primer SEQ ID NO:PCR ConditionsWatson-Crick Tra ns- versions non- Watson-Crick Trans- version s Tran- sitions GGGTGATTGAAGTTTGCTCCAGGGGGTGATTGAAGTTTGCTGCAGG (SEQ ID NO:424)ACGGATTCACGACGGAGGTGC 462 . CCGAGTCCGTGGCAGAGAGC 774. DMSO 0/1 2 2 TGTGGTTGAAGTAGGGGACAGGT 463.TGGCCCAATTGGAAGTGATTTC GT775. DMSO 3 1 0TGGGATGGCAGAGTCATCAACGT 464 . GGCCCAATCGGTAGAGGATGCA 776. DMSO 0 3 1ATGGGGCGCTCCAGTCTGTG 465. TGCACCCACACAGCCAGCAA 777. DMSO 0 3 1GGGGAGGGAGGACCAGGGAA 466. AATTAGCTGGGCGCGGTGGT 778.72C Anneal, 3% DMSO1 3ATCCCGTGCAGGAAGTCGCC 467. CAGGCGGCCCCTTGAGGAAT 779. DMSO 3 1 0CCCCAACCCTTTGCTCAGCG 468. TGAGGAGAACACCACAGGCAGA 780. DMSO 1 2 1ATCGACGAGGAGGGGGCCTT 469. CCCCTCACTCAAGCAGGCCC 781. DMSO 0 3 1TGCTCAAGGGGCCTGTTCCA 470 . CAGGGGCAGTGGCAGGAGTC 782. No DMSO 1 3 0TGCCTGGCACGCAGTAGGTG 471. GGGAAGGGGGAACAGGTGCA 783. DMSO 0 0 5Not optimized 1 1 3ACCTGGGCTTGCCACTAGGG 472 . GCTGCTCGCAGTTAAGCACCA 784. DMSO 1 3 1GTGGCCGGGCTACTGCTACC 473. GGTTCCACAAGCTGGGGGCA 785. DMSO 3 2 0Not optimized 1 3 1GCAAGAGGCGGAGGAGACCC 474.AGAGTCATCCATTTCCTGGGGG C786. DMSO 2 3 0GGGGTCAGTGGTGATATCCCCCT 475.AGGGAATCCTTTTTCCATTGCTTGTTT787.IM betaine, TD4 0AGAGAGGCCACGTGGAGGGT 476. GCCTCCCCTCCTCCTTCCCA 788. DMSO 1 3 1GACAGTGCCTTGCGATGCAC 477 . TCTGACCGGTATGCCTGACG 789. DMSO 3 2 0TGTGTGAACGCAGCCTGGCT 478.TGGTCTAGTACTTCCTCCAGCC TT790. DMSO 3 1 1GGTTCTCCCTTGGCTCCTGTGA 479. CCCACTGCTCCTAGCCCTGC 791. DMSO 1 3 1TGAAGTCAACAATCTAAGCTTCCACCT 480.AGCTTTGGTAGTTGGAGTCTTTGAAGG792. DMSO 3 1 2TGATTGGGCTGCAGTTCATGTACA 481. GCACAGCCTGCCCTTGGAAG 793. DMSO 2 1 3TCCATGGGCCCCTCTGAAAGA 482. AGCGGCTTCTGCTTCTGCGA 794. DMSO 1 0 5 W O 2014/144592 . o PC T/U S2014/029068 TABLE B Actual Target in U2OS.EGFP cells Forward PCR Primer SEQ ID NO Reverse PCR Primer SEQ IDNO:PCR ConditionsWatson- Crick Trans- versions non- Watson -Crick Trans- version s Tran- sitions GCGGTTGGTGGGGTTGATGC 483. GAGTTCCTCCTCCCGCCAGT 795. DMSO 2 0 4AGGCAAGATTTTCCAGTGTGCAAGA 484 . GCTTTTGCCTGGGACTCCGC 796. DMSO 2 0 4GCTGCTGGTCGGGCTCTCTG 485. GCTCTGTCCCACTTCCCCTGG 797. No DMSO TD 3 1 2GCTGCGAGGCTTCCGTGAGA 486. CGCCCCTAGAGCTAAGGGGGT 798. DMSO 3 2 1CCAGGAGCCTGAGAGCTGCC 487. AGGGCTAGGACTGCAGTGAGC 799. DMSO 1 3 2CTGTGCTCAGCCTGGGTGCT 488. GCCTGGGGCTGTGAGTAGTTT 800. DMSO 2 3 1AGCTCGCGCCAGATCTGTGG 489. ACTTGGCAGGCTGAGGCAGG 801.72C Anneal, 3% DMSO2 0 A f f V* TAI ״ A f' ، A If A * f f rn» r״r*T 803 GTCCAGTGCCTGACCCTGGC 493. AGCATCATGCCTCCAGCTTCA 80S. DMSO 1 1 1GCTCCCGATCCTCTGCCACC 494. GCAGCTCCCACCACCCTCAG 806. DMSO 1 2 0GGGGACAGGCAGGCAAGGAG 495. GTGCGTGTCCGTTCACCCCT 807. DMSO 1 1 1AAGGCGCIGCICCGTAGGAC - * *GACCCTCAGGAAGCTGGGAG 497. CTGCGAGATGCCCCAAATCG 809.IM betaine, TD0 2CCGCGGCGCTCTGCTAGA 498. TGCTGGGATTACAGGCGCGA 810. DMSO 1 1 1CCAGGTGGTGTCAGCGGAGG،،؛TGCCTGGCCCTCrCTGAGTCT 811 VCGACTCCACG G CGTCTCAG G 500. CAG CG CAGTCCAG CCCG ATG 812.IM betaine, TD1 0CTTCCCTCCCCCAGCACCAC 501.GCTACAGGTTGCACAGTGAGAGGT813. DMSO 1 1 1CCCCGGGGAGTCTGTCCTGA 502. CCCAGCCGTTCCAGGTCTTCC 814.72C Anneal, 3% DMSO0 2GAAGCGCGAAAACCCGGCTC 503. TCCAGGGTCCTTCTCGGCCC 815. DMSO 1 0 2AGGGTGGTCAGGGAGGCCTT 504. CATGGGGCTCGGACCTCGTC 816. DMSO 2 0 1GGGAAGAGGCAGGGCTGTCGWHBTGCtAGCAAGGAAGCTGGCC ،|||،72C Aanau.t,־ W O 2014/144592 ... PC T/U S2014/029068 TABLE B Actual Target in U2OS.EGFP cells Forward PCR Primer SEQ ID NO Reverse PCR Primer SEQ ID NO:PCR ConditionsWatson- Crick Trans- versions non- Watson -Crick Trans- version s Tran- sitions GAGTGACGATGAGCCCCGGG 506. CCCTTAGCTGCAGTCGCCCC 818.68C Anneal, 3% DMSO1 3 V ،،،CACCTGGGGCATCTGGGTGG 508. ACTGGGGTTGGGGAGGGGAT 820. DMSO 2 0 2K'J. #88*^■11■؛،،؟ AGGAAATGIG- IGIGCLAGGGC DMSO ؛،،،GCCTCAGACAACCCTGCCCC 511.GCCAAGTGTTACTCATCAAGAAAGTGG823. No DMSOTD 2 1 1GCCGGGACAAGACTGAGTTGGG 512. TCCCGAACTCCCGCAAAACG 824. DMSO 1 2 1TGCTGCAGG/GI ILLUGAG،،،ACACTGGTCCAGGTCCCGTCf■®■I■CTCTCCCCCCACCCCCCCTCTGG(SEQ ID NO:425)ATCGCGCCCAAAGCACAGGT 515.AGGCTTCTGGAAAAGTCCTCAATGCA827. DMSO 3 0 2Not optimized 1 1 2CCCTCATG GTG GTCTTACG G C A 516. AG CCACAC ATCTTTCTG GT AG G G 828. DMSO 1 1 2TGCGTCGCTCATGCTGGGAG 517. AGGGTGGGGTGTACTGGCTCA 829. DMSO 0 3 1GAGCTGAGACGGCACCACTG|،|g| BBHB،■!lllllllllNot optimized 1 2iAGTGAGAGTGGCACGAACCA 519. CAGTAGGTGGTCCCTTCCGC 831. DMSO 2 1 1Not optimized 832. 1 1 3GGGAGAACCTTGTCCAGCCT 520. AAGCCGAAAAGCTGGGCAAA 833. DMSO 0 2 3־ - 1 1Not optimize□ 1 0 4CTGAGAGGGGGAGGGGGAGG 522. TCGACTGGTCTTGTCCTCCCA 835.68C Anneal, 3% DMSO0 2CAGCCTGCTGCATCGGAAAA 523. TGCAGCCAAGAGAAAAAGCCT 836.IM betaine, TD0 4TCCCTCTGACCCGGAACCCA 524. ACCCGACTTCCTCCCCATTGC 837. DMSO 2 1 2TGGGGGTTGCGTGCTTGTCA 525. GCCAGGAGGACACCAGGACC 838. DMSO 4 1 0ATCAGGTGCCAGGAGGACAC 526. GGCCTGAGAGTGGAGAGTGG 839. DMSO 4 1 0 W O 2014/144592 cn PCT/US2014/029068 TABLE B Actual Target in U20S.EGFP cells Forward PCR PrimerSEQ ID NOReverse PCR PrimerSEQ IDNO:PCR ConditionsWatson- Crick Trans- versions non- Watson -Crick Trans- version s Tran- sitions Not optimized4 0TGAGCCACATGAATCAAGGCCTCC 527.ACCTCTCCAAGTCTCAGTAACTCTCT840. DMSO 1 3 1GGTCCCTCTGTGCAGTGGAA 528. CTTTG GTG G ACCTG CAC AG C 841. DMSO 2 2 2GCGAGGCTGCTGACTTCCCT 529. GCTGGGACTACAGACATGTGCCA 842. DMSO 2 2 2ATTTCCTCCCCCCCC-CCTCAGG(SEQ ID NO:426)ATTGCAGGCGTGTCCAGGCA 530. AAATCCTGCATGGTGATGGGAGT 843. DMSO 1 1 5TGCTCTGCCATTTATGTCCTATGAACT 531. ACAGCCTCTTCTCCATGACTGAGC 844. DMSO 1 3 2TCCGCCCAAACAGGAGGCAG 532. GCGGTGGGGAAGCCATTGAG 845. DMSO 2 3 1GGGGGTCTGGCTCACCTGGA 533. CCTGTCGGGAGAGTGCCTGC 846. DMSO 3 1 2TCCTG GTTCATTTG CTAG AACTCTG GA 534. ACTCCAGATGCAACCAGGGCT 847. DMSO 3 2 1CGTGTGGTGAGCCTGAGTCT 535. GCTTCACCGTAGAGGCTGCT 848. DMSO 3 0 3AG G CCCTG ATAATTCATG CTACC AA 536.TCAGTGACAACCTTITGTATTCGGCA849. DMSO 0 2 4Not optimized2 2537.TCCAGATGGCACATTGTCAG 538. AGGGAGCAGGAAAGTGAGGT 850. DMSO■■ 851.; ATa-1. TACCCGGGCCGTCTGTAGA •‘s.GACACCCCACACACTCTCATGC 541. TGAATCCCTTCACCCCCAAG 853. DMSO 1 0 11.״I.':. • ' 1 .1CAGGGCCAGGAACACAGGAA 543. GGGAGGTATGTGCGGGAGTG 855. DMSO 1 1 0TGCAGCCTGAGTGAGCAAGTGT 544. GCCCAGGTGCTAAGCCCCTC 856. DMSO 1 0 1TACAGCCTGGGTGATGGAGC 545. TGTGTCATGGACTTTCCCATTGT 857.IM betaine, TD1 0GGCAGG CATTA AACTC ATC AGGTCC 546. TCTCCCCCAAGGTATCAGAGAGCT 858. DMSO 1 1 0GGGCCTCCCIXit i'GG1I'CTC GCTGCCGTC.CGAACCCAaGAACAAACGCAGGTGGACCGAA 548. ACTCCGAAAATGCCCCGCAGT 860. DMSO 1 1 0AGGGGAGGGGACATTGCCT 549. TTGAGAGGGTTCAGTGGTTGC 861. DMSO 1 0 1CTA ATGCTTACG G CTG CGG G 550. AGCCAACGGCAGATGCAAAT 862. DMSO 1 0 1 W O 2014/144592 cd PCT/US2014/029068 TABLE B Actual Target in U20S.EGFP cellsForward PCR Primer SEQID NO Reverse PCR PrimerSEQIDNO:PCR ConditionsWatson- Crick Trans- versions non- Watson -Crick Trans- version s Tran- sitions GAGCGAAGTTAACCCACCGC 551. CACACATGCACATGCCCCTG 863.68C, 3% DMSO0 0GCATGTGTCTAACTGGAGACAATAGCA 552. TCCCCCATATCAACACACACA 864. DMSO 2 0 0GCCCCTCCCGCCI 1 1 IGIGT 553.TGGGCAAAGGACATGAAACAGAC A865. DMSO 2 0 0 GCCTCAGCTCTGCTCTTAAGCCC 554.ACGAACAGATCAI1 1 1 ICATGGCT TCC866. DMSO 2 0 0■HillTCTGTCACCACACAG 1 ؛ACCACC 868■■■GGGGACCCTCAAGAGGCACT 557. GGGCATCAAAGGATGGGGAT 869. DMSO 2 0 1؛،، a: CGGGGTGGCAGTGACGTCAA 559. GGTGCAGTCCAAGAGCCCCC 871. DMSO 0 0 3AGCTGAGGCAGAGTCCCCGA 560. GGGAGACAGAGCAGCGCCTC 872. DMSO 1 1 1ACCACCAGACCCCACCTCCA 561. AGGACGACTTGTGCCCCATTCA 873.72C Anneal, 3% DMSO1 1GGGTCAGGACGCAGGTCAGA 562. TCCACCCACCCACCCATCCT 874.72C Anneal, 3% DMSO0 1ACACTCTGGGCTAGGTGCTGGA 563. GCCCCCTCACCACATGATGCT 875. DMSO 2 0 1GG G G CC ATTCCTCTG CTGC A 564. TGGGGATCCTTGCTCATGGC 876. DMSO 3 0 0ACACACTGGCTCGCATTCACCA 565. CCTGCACGAGGCCAGGTGTT 877. DMSO 2 1 0TGGGCACGTAGTAAACTGCACCA 566. CTCGCCGCCGTGACTGTAGG 878. DMSO 0 3 1TCAGCTGGTCCTGGGCTTGG 567. AGAGCACTGGGTAGCAGTCAGT 879. DMSO 2 1 0AGACACAGCCAGGGCCTCAG 568. GGTGGGCGTGTGTGTGTACC 880.68C, 3% DMSO1 1ACACTCTCACACACGCACCAA 569. GAGAAGTCAGGGCTGGCGGG 881.72C Anneal, 3% DMSO2 0ACTGCCTGCATTTCCCCGGT 570. TGGTGAGGGCTTCAGGGAGC 882. DMSO 1 1 1GCCAGGTTCATTGACTGCCC 571. TCCTTCTACACATCGGCGGC 883. DMSO 2 1 0CGAGGGAGCCGAGTTCGTAA 572. CTGACCTGGGGCTCTGGTAC 884. DMSO 1 2 0TCCTCGGGAAGTCATG G CTTC A 573. GCACTGAGCAACCAGGAGCAC 885. DMSO 2 1 0Not optimized0 3TAAACCGTTGCCCCCGCCTC 574. GCTCCCCTGCCAGGTGAACC 886. DMSO 2 1 1 W O 2014/144592 cn PCT/US2014/029068 TABLE B Actual Target in U2OS.EGFP cellsForward PCR PrimerSEQID NOReverse PCR PrimerSEQIDNO:PCR ConditionsWatson- Crick Trans- versions non- Watson-Crick Tra ns- version s Tran- sitions CCTGCTGAGACTCCAGGTCC 575. CTGCGGAGTGGCTGGCTATA 887. DMSO 2 0 2CTCGGGGACTGACAAGCCGG 575. GGAGCAGCTCTTCCAGGGCC 888. DMSO 3 0 1CCCCGACCAAAGCAGGAGCA 577. CTGGCAGCCTCTGGATGGGG 889. DMSO 1 2 1Not optimized 0 3 1ATTTCAGAGCCCCGGGGAAA 578. AGGCCGCGGTGTTATGGTTA 890. DMSO 1 2 1GCCAGTGGCTTAGTGT 1 1 1G1 GT 579.TGACATATTTTCCTGGGCCATGGGT891. DMSO 2 1 1TGCCAGAAGAACATGGGCCAGA 580.CCATGCTGACATCATATACTGGGAAGC892. DMSO 3 1 0GCGTGTCTCTGTGTGCGTGC 581. CCAGGCTGGGCACACAGGTT 893. DMSO 3 1 0Not optimized 2 2 0TGCCCAGTCCAATATTTCAGCAGCT 582.AGGATGAGTTCATGTCU 1 1 1 b 1 bGGG894. DMSO 2 2 0GGGTGAAAATTTGGTACTGTTAGCTGT 583.AATGACTCATTCCCTGGGTATCTC CCA895. DMSO 2 2 0TG CCCCATC A ATC ACCTCG G C 584. CAAGGTCGGCAGGGCAGTGA 896. DMSO 1 2 2GCCTCCTCTGCCGCTGGTAA 585. TGAGAGTTCCTGTTGCTCCACACT 897. DMSO 1 2 2Not optimized 2 2 1GCCACCAAAATAGCCAGCGT 586. AC ATG C ATCTGTGTGTG CGT 898. DMSO 3 0 2ACAGACTGACCCTTGAAAAATACCAGT 587.TGTATLI ULI IGCCAATGGI 1 1 ICCC899. DMSO 2 1 2AGCCAAAI 1 1 LI LAACAGCAGCACT 588. TCCTGGAGAGCAGGCAI 1 1 1 IGI 900. DMSO 3 1 1ACCTCCTTGTGCTGCCTGGC 589. GGCGGGAAGGTAACCCTGGG 901. DMSO 2 1 2CACAAAGCTCTACCTTTCCAGTAGTGT 590. TGATCCGATGGTTGTTCACAGCT 902. DMSO 3 1 1TGTGGGGATTACCTGCCTGGC 591. ACGCACAAAAATGCCCTTGTCA 903. DMSO 2 2 1TGAGGCAGACCAGTCATCCAGC 592. GCCCGAGCACAGTGTAGGGC 904. DMSO 2 3 0ATTAGCTGGGCGTGGCGGAG 593. ACTGCATCTCATCTCAGGCAGCT 905. DMSO 2 1 3TGAAGCAGAAGGAGTGGAGAAGGA 594.TCAGCTTCACATLlbl1 ICAGTTC AGT906. DMSO 4 0 2TGGTGGAGTGTGTGTGTGGT 595. AGAGCAGAAAGAGAGTGCCCA 907. DMSO 1 3 2GCCCCTGTACGTCCTGACAGC 596. TGCACAAGCCACTTAGCCTCTCT 908. DMSO 3 1 2AGCGCAGGTAAACAGGCCCA 597. TCTCTCGCCCCGTTTCCTTGT 909. DMSO 3 1 2 W O 2014/144592 co PCT7US2014/029068 TABLE B Actual Target in U20S.EGFP cells Forward PCR Primer SEQ ID NO Reverse PCR Primer SEQ ID NO:PCR ConditionsWatson- Crick Trans- versions non- Watson -Crick Trans- version s Tran- sitions ATGGGTGCCAGGTACCACGC 598. ACAGCAGGAAGGAGCCGCAG 910. DMSO 2 3 1CGGGCGGGTGGACAGATGAG 599. AGGAGGTCTCGAGCCAGGGG 911. DMSO 2 3 1TCAACCTAGTGAACACAGACCACTGA 600.GTCTATATACAGCCCACAACCTCATGT912. DMSO 1 2 3GCCAGGGCCAGTGGATTGCT 601.TGTCAI 1 ILI 1AGTATGTCAGCCG GA913. DMSO 2 4 0GAGCCCCACCGGTTCAGTCC 602. GCCAGAGCTACCCACTCGCC 914. DMSO 1 3 2603.GGAGCAGCTGGTCAGAGGGG 604. GGGAAGGGGGACACTGGGGA 915. DMSO T. ؛ (J CC1tCAACK Al GAC,CAG(aIliillllATCTGCACATGTATGTACAGGAG1AAGACAGAGGAGAAGAAGAAGGG (SEQ ID NO:427)TGGGGAATCTCCAAAGAACCCCC 606.AGGGTGTACTGTGGGAACTTTGC A917. DMSO 2 1 1 GATGGCCCCACTGAGCACGT 607.ACTTCGTAGAGCCTTAAACATGTGGC918. DMSO 1 0 2AGGATTAATGTTTAAAGTCACTGGTGG 608. TCAAACAAGGTGCAGATACAGCA 919.IM betaine, TD0 2TCCAAGCCACTGGI i ICICAGTCA 609.TGCTCTGTGGATCATATTTTGGGG GA920. DMSO 0 1 2ACTTTCAGAGCTTGGGGCAGGT 610. CCCACG CIG A AGTG CAATGG C 921. DMSO 1 1 1CAAAGCATGCCTTTCAGCCG 611. GGCTCTTCGATTTGGCACCT 922.IM betaine, TD1 1Not optimized 1 0 2GGACTCCCTGCAGCTCCAGC 612. AGGAACACAGGCCAGGCTGG 923.72C Anneal, 6% DMSO0 3CCCTTAGGCACCTTCCCCA 613. CCGACCTTCATCCCTCCTGG 924. DMSO 0 1 2TGATTCTGCCTTAGAGTCCCAGGT 614. TGGGCTCTGTGTCCCTACCCA 925. DMSO 0 3 0Not optimized 2 1 0AGGCAGGAGAGCAAGCAGGT 615. ACCCTGACTACTGACTGACCGCT 926. DMSO 0 1 2CTCCCCATTGCGACCCGAGG 616. AGAGGCATTGACTTGGAGCACCT 927. DMSO 1 2 0CTGGAGCCCAGCAGGAAGGC 617. CCTCAGGGAGGGGGCCTGAT 928. DMSO 1 2 0 W O 2014/144592 c , PCT/US2014/029068 TABLE B Actual Target in U20S.EGFP cellsForward PCR PrimerSEQID NOReverse PCR PrimerSEQ ID NO:PCR ConditionsWatson- Crick Tra ns- versions non- Watson -Crick Trans- version s Tran- sitions ACTGTGGGCGTTGTCCCCAC 618. AGGTCGGTGCAGGGTTTAAGGA 929. DMSO 1 0 3GGCGCTCCLIIIIIUCIIIGI 619. CGTCACCCATCGTCTCGTGGA 930. DMSO 2 0 2TGCCATCTATAGCAGCCCCCT 620.GCATCTTGCTAACCGTACTTCTTC TGA931. DMSO 1 0 3GTGGAGACGCTAAACCTGTGAGGT 621. GCTCCTGGCCTCTTCCTACAGC 932. DMSO 1 2 1CCGAACTTCTGCTGAGCTTGATGC 622. CCAAGTCAATGGGCAACAAGGGA 933. DMSO 0 2 2Not optimized-1 2TGCCCCCAAGACLI 1 ILlUL 623. ATGGCAGGCAGAGGAGGAAG 934. DMSO 2 0 2GGGTGGGGCCATTGTGGGTT 624. CTGGGGCCAGGGTTTCTGCC 935. DMSO 3 0 1TGGAGAACATGAGAGGCTTGCAA 625.TCCTTCTGTAGGCAATGGGAACA A936. DMSO 3 0 1GCCACATGGTAGAAGTCGGC 626. GGCAGATTTCCCCCATGCTG 937.IM betaine, TD2 1TGTACACCCCAAGTCCTCCC 627. AAGGGGAGTGTGCAAGCCTC 938. DMSO 3 1 0AGGTCTGGCTAGAGATGCAGCA 628.AGTCCAACACTCAGGTGAGACCCT939. DMSO 3 1 0CCAAGAGGACCCAGCTGTTGGA 629.GGGTATGGAATTCTGGATTAGCAGAGC940. DMSO 0 2 2ACCATCTCTTCATTGATGAGTCCCAA 630. ACACTGTGAGTATGCTTGGCGT 941. DMSO 2 2 0GGCTGCGGGGAGATGAGCTC 631. TCGGATGCTTTTCCACAGGGCT 942. DMSO 2 2 1TCTTCCAGGAGGGCAGCTCC 632. CCAATCCTGAGCTCCTACAAGGCT 943. DMSO 1 0 4GAGCTGCACTGGATGGCACT 633. TGCTGGTTAAGGGGTGTTTTGGA 944. DMSO 1 1 3TCTGGGAAGGTGAGGAGGCCA 634.TGGGGGACAATGGAAAAGCAATGA945. DMSO 0 2 3CTTGCTCCCAGCCTGACCCC 635. AG CCCTTG CC ATG CAG G ACC 946. DMSO 3 1 1GGGAI 1 1 1 IAICIGTTGGGTGCGAA 636. AACCACAGATGTACCCTCAAAGCT 947. DMSO 2 2 1ACCCATCAGGACCGCAGCAC 637. TCTGGAACCTGGGAGGCGGA 948.72C Anneai, 3% DMSO1 1CGTCCCTCACAGCCAGCCTC 638. CCTCCTTGGGCCTGGGGTTC 949. DMSO 1 3 1CCCTCTGCAAGGTGGAGTCTCC 639. AGATGTTCTGTCCCCAGGCCT 950. DMSO 1 3 1GGCTTCCACTGCTGAAGGCCT 640. TGCCGCTCCACATACCCTCC 951. DMSO 2 1 2 W O 2014/144592 c c PCT/US2O14/029068 TABLE B Actual Target in U20S.EGFP cells Forward PCR Primer SEQ ID NO Reverse PCR Primer SEQ ID NO: PCR Conditions Watson- Crick Trans- versions non- Watson-Crick Trans- version s Tran- sitions AGCATTGCCTGTCGGGTGATGT 641.AGCACCTATTGGACACTGGTTCTCT952. DMSO 1 3 1TCTAGAGCAGGGGCACAATGC 642. TGGAGATGGAGCCTGGTGGGA 953. DMSO 2 2 1GGTCTCAGAAAATGGAGAGAAAGCACG 643. CCCACAGAAACCTGGGCCCT 954. DMSO 1 2 3GGTTGCTGATACCAAAACGTTTGCCT 644. TGGGTCCTCTCCACCTCTGCA 955. DMSO 0 3 3ACTCTCCTTAAGTACTGATATGGCTGT 645. CAGAATCTTGCTCTGTTGCCCA 956. DMSO 0 4 2Not optimized2 2Not optimized2 2CAATGCCTGCAGTCCTCAGGA 646.TCCCAAGAGAAAACTCTGTCCTGACA957. DMSO 4 1 1GCATTGGCTGCCCAGGGAAA 647. TGGCTGTGCTGGGCTGTGTT 958. DMSO 2 2 2CCACAAGCCTCAGCCTACCCG 648. ACAGGTGCCAAAACACTGCCT 959. DMSO 2 1 3TCATTGCAGCAGAAGAAGAAAGGTCATTGTAGCAGAAGAAGAAAGG(SEQ ID NO:428)GCCTCTTGCAAATGAGACTCCTTTT 649. CGATCAGTCCCCTGGCGTCC 960. DMSO 2/1 2/3 2 TCCCAGAATCTGCCTCCGCA 650. AGGGGTTTCCAGGCACATGGG 961. DMSO 0 4 2651. 962.TCCTAAAAATCAGTTTTGAGATTTACTTCC 652.AAAGTGTTAGCCAACATACAGAAGTCAGGA963. DMSOGGTATCTAAGTCATTACCTGTGGGGTATCTAAGTCAATACCTGTGG(SEQ ID NO:429)ACATCTGGGGAAAGCAAAAGTCAACA 653.TGTCTGAGTATCTAGGCTAAAAGTGGT964. DMSO 1/2 1 1 ACGATCTTGCTTCATTTCCCTGTACA 654.AGTG CTTTGTG A ACTG AA A AG CA AACA965. DMSO 0 3 0 GCACCTTG GTG CTG CTAAATG CC 655.G G G CAACTG AACAG G CATG AATGG966. DMSO 1 2 0AACTGTCCTGCATCCCCGCC 656. GGTGCACCTGGATCCACCCA 967. DMSO 1 1 1Not optimized1 1 W O 2014/144592 c c PCT/US2014/029068 TABLE B Actual Target inU2OS.EGFP cells Forward PCR Primer SEQ ID NO Reverse PCR Primer SEQ ID NO:PCR ConditionsWatson- Crick Trans- versions non- Watson -Crick Trans- version s Tran- sitions CATCACCCTCCACCAGGCCC 657. ACCACTGCTGCAGGCTCCAG 968.72C Anneal, 3% DMSO3 0Not optimized 2 0 2CCTGACCCGTGGTTCCCGAC 658. TGGTGCGTGGTGTGTGTGGT 969.72C Anneal, 3% DMSO2 1TGGGAACATTGGAGAAGTTTCCTGA 659. CCATGTGACTACTGGGCTGCCC 970. DMSO 1 1 2AGCCTTGGCAAGCAACTCCCT 660.GGTTCTCTCTCTCAGAAAAGAAA GAGG 971. DMSO 1 0 3GGCAGCGGACTTCAGAGCCA 661. GCCAGAGGCTCTCAGCAGTGC 972. DMSO 1 0 3CCAGCCTGGTCAATATGGCA 662. ACTGTGCCCAGCCCCATATT 973. DMSO 2 1 1ATGCCAACACTCGAGGGGCC 663. CGGGTTGTGGCACCGGGTTA 974. DMSO 2 1 1TTGCTCTAGTGGGGAGGGGG 664.AGAGTTCAGGCATGAAAAGAAGC AACA975. DMSO 3 0 1AGCTGAAGATAGCAGTGTTTAAGCCT 665. TGCAATTTGAGGGGCTCTCTTCA 976. DMSO 1 1 2AGTCACTGGAGTAAGCCTGCCT 666.TGCCAGCCAAAAGTTGTTAGTGTGT977. DMSO 2 0 2 GGGTCTCCCTCAGTGCCCTG 667.TGTGTGGTAGGGAGCAAAACGAC A978. DMSO 2 0 2TGGGGGCTGTTAAGAGGCACA 668. TGACCACACACACCCCCACG 979. DMSO 1 2 1TCAAAACAGATTGACCAAGGCCAAAT 669. Tb1G1 1 1 1 1 AAGCTGCACCCCAGG 980. DMSO 1 0 3TCTGGCACCAGGACTGATTGTACA 670. GCACGCAGCTGACTCCCAGA 981. DMSO 1 2 1Not optimized 1 0 3AGCATCTGTGATACCCTACCTGTCT 671. ACCAGGGCTGCCACAGAGTC 982. DMSO 1 0 3TAGTCTTGTTGCCCAGGCTG 672. CTCGGCCCCTGAGAGTTCAT 983. DMSO 1 2 1TCCATCTCACTCATTACCTGAGGTCCATCTCACTCATTACCTGATG(SEQ ID NO:430)CTGCAACCAGGGCCCTTACC 673. GAGCAGCAGCAAAGCCACCG 984. DMSO 1 1 2 GCCTGGAGAGCAAGCCTGGG 674. AGCCGAGACAATCTGCCCCG 985. DMSO 1 1 2TTTATATTAGTGATTACCTGCGG (SEQ ID NO:431)AGTG A AAC A A ACAAG C AG C AGTCTG A 675. GGCAGGTCTGACCAGTGGGG 986. No DMSOTD 1 2 1 W O 2014/144592 ״ PCT/US2014/029068 TABLE B Actual Target in U2OS.EGFP cellsForward PCR PrimerSEQ1D NOReverse PCR PrimerSEQIDNO:PCR ConditionsWatson- Crick Trans- versions non- Watson -Crick Trans- version s Tran- sitions AGGCTCAGAGAGGTAAGCAATGGA 676.TGAGTAGACAGAAATGTTACCGG TGTT987. DMSO 3 0 2TCAGAGATGTTAAAGCCTTGGTGGG 677. AGTGAACCAAGGGAATGGGGGA 988. DMSO 3 0 2TGTG CTTTCTGG G GTAGTG G C A 678. CACCTCAGCCCTGTAGTCCTGG 989. DMSO 0 4 1CCATTGGGTGACTGAATGCACA 679. GCCACTGTCCCCAGCCTATT 990.IM betaine, TD3 1ACCAAGAAAGTGAAAAGGAAACCC 680. TGAGATGGCATACGATTTACCCA 991. DMSO 1 2 2AGGGTGGGGACTGAAAGGAGCT 681.TGGCATCACTCAGAGATTGGAACACA992. DMSO 3 1 1ACCAGTGCTGTGTGACCTTGGA 682. TCCTATGGGAGGGGAGGCTTCT 993. DMSO 3 1 1CCAGGTGTG GTG GTTCATG AC 683. GCATACGGCAGTAGAATGAGCC 994.68C, 3% DMSO0 1CAG G CGCTGG GTTCTT AG CCT 684. CCTTCCTG G G CCCC ATG GTG 995. DMSO 2 3 0TGGGGTCCAAGATGTCCCCT 685.TGAAACTGCTTGATGAGGTGTGG A996. DMSO 1 2 2GCTGGGCTTGGTGGTATATGC 686.ACTTGCAAAGCTGATAACTGACT GA997. DMSO 5 0 1AGTTGGTGTCACTGACAATGGGA 687. CGCAGCGCACGAGTTCATCA 998. DMSO 3 0 3AGAGGAGGCACAATTCAACCCCT 688. GGCTGGGGAGGCCTCACAAT 999. DMSO 1 1 4GGGAAAGTTTGGGAAAGTCAGCA 689. AGGACAAGCTACCCCACACC 1000. DMSO 1 3 2TG GTGCATC A A AG G GTTGCTTCT 690. TCATTCCAGCACGCCGGGAG 1001. DMSO 0 3 3CCCAGGCTGCCCATCACACT 691.TGGAGTAAGTATACCTTGGGGACCT1002. DMSO 1 3 2TCAGTGCCCCTGGGTCCTCA 692. TGTGCAAATACCTAGCACGGTGC 1003. DMSO 4 2 0AGCACTCCCTTTTGAATTTTGGTGCT 693.ACTGAAGTCCAGCCTCTTCCATTT CA1004. DMSO 2 1 3GAAACCGGTCCCTGGTGCCA 694.GGGGAGTAGAGGGTAGTGTTGCC1005. DMSO 2 0 4TTGCGGGTCCCTGTGGAGTC 695. AGGTGCCGTGTTGTGCCCAA 1006. DMSO 1 2 3696. 1007.GCCCTACATCTGCTCTCCCTCCA 697. G G G CCG G G AAAG AGTTG CTG 1008. DMSOTTGGAGTGTGGCCCGGGTTG 698. ACCILILI 1 IUIUlGCCTCACTGT 1009. DMSO 0 1 1 W O 2014/144592 EO PC T/U S2014/029068 TABLE B Actual Target in U20S.EGFP cells Forward PCR Primer SEQ ID NO Reverse PCR Primer SEQ ID NO:PCR ConditionsWatson- Crick Trans- versions non- Watson -Crick Trans- version s Tran- sitions CACACCATGCTGATCCAGGC 699. GCAGTACGGAAGCACGAAGC 1010. DMSO 1 1 1CTCCAGGGCTCGCTGTCCAC 700. CTGGGCTCTGCTGGTTCCCC 1011. DMSO 0 2 1CTGTGGTAGCCGTGGCCAGG 701. CCCCATACCACCTCTCCGGGA 1012. DMSO 0 2 1GGTGGCGGGACTTGAATGAG 702. CCAGCG 1 b 1 i 1CCAAGGGAT 1013.IM betaine, TD1 2GGAATCCCCTCTCCAGCCCCTGGGGAATCCCCTCTCCAGCCTCTGG(SEQ ID NO:432)CCAGAGGTGGGGCCCTGTGA 703. TTTCCACACTCAGTTCTGCAGGA 1014. DMSO 1 1 1/2 GGAATCTCTTCCTTGGCATCTGG(SEQ ID NO:433)TGTGACTGGTTGTCCTGCTTTCCT 704. G C AGTGTTTTGTGGTG ATGG G C A 1015.IM betaine, TD1 5CTGGCCAAGGGGTGAGTGGG 705. TGGGACCCCAGCAGCCAATG 1016. DMSO 1 0 2ACGGTGTGCTGGCTGCTCTT 706. ACAGTGCTGACCGTGCTGGG 1017. DMSO 1 1 1TGGTTTGGGCCTCAGGGATGG 707. TGCCTCCCACAAAAATGTCTACCT 1018. DMSO 0 0 3TGGTTTGGGCCTCAGGGATGG 708. ACCCCTTATCCCAGAACCCATGA 1019. DMSO 0 0 3TCCAAGTCAGCGATGAGGGCT 709. TGGGAGCTGTTCLI 1 1 1 IGGCCA 1020. DMSO 0 3 0CACCCCTCTCAGCTTCCCAA 710. GCTAGAGGGTCTGCTGCCTT 1021. DMSO 1 2 0AGACCCCTTGGCCAAGCACA 711. CTTGCTCTCACCCCGCCTCC 1022. DMSO 2 1 0ACATGTGGGAGGCGGACAGA 712.TCTCACTTTGCTGTTACCGATGTCG1023. DMSO 0 1 3GGACGACTGTGCCTGGGACA 713.AGTGCCCAGAGTGTTGTAACTGCT1024.72C Anneal, 3% DMSO1 3G G AG AG CTC AGCG CCA G GTC 714. CAGCGTGGCCCGTGGGAATA 1025. DMSO 1 1 2GCTGAAGTGCTCTGGGGTGCT 715.ACCCCACTGTGGATGAATTGGTACC1026. DMSO 1 1 2TCGGGGTGCACATGGCCATC 716. TTGCCTCGCAGGGGAAGCAG 1027. DMSO 0 1 3CTCGTGGGAGGCCAACACCT 717. AGCCACCAACACATACCAGGCT 1028. DMSO 2 0 2GCATGCCTTTAATCCCGGCT 718. AGGATTTCAGAGTGATGGGGCT 1029. DMSO 2 1 1CGCCCAGCCACAAAGTGCAT 719.GCAAA HILI GCACCTACTCTAGGCCT1030. DMSO 1 1 2 W O 2014/144592 cr1 PCT/US2014/029068 TABLE B Actual Target in U20S.EGFP cells Forward PCR Primer SEQ ID NO Reverse PCR Primer SEQ ID NO:PCRConditionsWatson- Crick Trans- versions non- Watson -Crick Trans- version s Tran- sitions AGCTCACAAGAATTGGAGGTAACAGT 720. GCAGTCACCCTTCACTGCCTGT 1031. DMSO 1 1 2AAACTGGGCTGGGCTTCCGG 721. GGGGCTAAGGCATTGTCAGACCC 1032. DMSO 2 0 2GCAGGTAGGCAGTCTGGGGC 722. TCTCCTGCCTCAGCCTCCCA 1033.IM betaine, TD2 1GCAGGTAGGCAGTCTGGGGC 723. TCTCCTGCCTCAGCCTCCCA 1034.IM betaine, TD2 1GCAGGTAGGCAGTCTGGGGC 724. TCTCCTGCCTCAGCCTCCCA 1035.IM betaine, TD2 1GCTCTGGGGTAGAAGGAGGC 725. GGCCTGTCAACCAACCAACC 1036. DMSO 2 2 0TGACATGTTGTGTGCTGGGC 726. A AATCCTG CAG CCTCCCCTT 1037. DMSO 0 2 2TCCTGGTGAGATCGTCCACAGGA in. TCCTCCCCACTCAGCCTCCC 1038. DMSO 0 3 1TCCTAATCCAAGTCL HIGH CAGACA 728. AGGGACCAGCCACTACCCTTCA 1039. DMSO 2 2 0GGGACACCAGTTCCTTCCAT 729. GGGGGAGATTGGAGTTCCCC 1040. DMSO 1 0 4ACACCACTATCAAGGCAGAGTAGGT 730. TCTGCCTGGGGTGCTTTCCC 1041. DMSO 1 1 3CTGGGAGCGGAGGGAAGTGC 731. GCCCCGACAGATGAGGCCTC 1042. DMSO 1 2 2CAGATTACTGCTGCAGCACCGGG(SEQ ID NO:434)CGGGTCTCGGAATGCCTCCA 732. ACCCAGGAATTGCCACCCCC 1043. DMSO 1 2 3TTGCTGTGGTCCCGGTGGTG 733. GCAGACACTAGAGCCCGCCC 1044. DMSO 3 2 0GGTGTGGTGACAGGTCGGGT 734. ACCTGCGTCTCTGTGCTGCA 1045. DMSO 2 3 0CTCCCAGGACAGTGCTCGGC 735. CCTGG CCCC ATG CTG CCTG 1046. DMSO 2 2 1TGCGTAGGTTTTGCCTCTGTGA 736. AGGGAATGATGTTTTCCACCCCCT 1047. DMSO 2 3 0CTCCGCAGCCACCGTTGGTA 737. TGCATTGACGTACGATGGCTCA 1048. DMSO 1 3 1ACCTGCAGCATGAACTCTCGCA 738.ACCTGAGCAACATGACTCACCTG G1049. DMSO 2 1 2ACACAAACTTCTGCAGCACCTGGACACAAACTTCTGCAGCACGTGG(SEQ ID NO:435)TCTCCAGTTTCTTGCTCTCATGG 739. ACCATTGGTGAACCCAGTCA 1050.IM betaine, TD3/2 3 1 TGGGGTGGTGGTCTTGAATCCA 740.TCAGCTATAACCTGGGACTTGTGC T1051. DMSO 2 1 3 W O 2014/144592 PC T/U S2014/029068 TABLE B Actual Target in U20S.EGFP cells Forward PCR Primer 5EQ ID NO Reverse PCR Primer SEQ ID NO:PCR ConditionsWatson- Crick Trans- versions non- Watson -Crick Trans- version s Tran- sitions AGCAGCCAGTCCAGTGTCCTG 741. CCCTTTCATCGAGAACCCCAGGG 1052. DMSO 3 1 2TGGACGCTGCTGGGAGGAGA 742. GAGGTCTCGGGCTGCTCGTG 1053. DMSO 0 3 3AGGTTTGCACTCTGTTGCCTGG 743. TGGGGTGATTGGTTGCCAGGT 1054. DMSO 3 2 1TCTTCCTTTGCCAGGCAGCACA 744.TGCAGGAATAGCAGGTATGAGGA GT1055. DMSO 4 0 2GGACGCCTACTGCCTGGACC 745. G CCCTG GCAGCCCATGGTAC 1056. DMSO 3 0 3AGGCAGTCATCGCCTTGCTA 746. GGTCCCACCTTCCCCTACAA 1057. DMSO 2 3 1Not optimized 3 1 2CCCCAGCCCCCACCAGTTTC 747. CAGCCCAGGCCACAGCTTCA 1058. DMSO 1 4 1 Sequences and characteristics of genomic on- and off-target sites for six RGNs targeted to endogenous human genes and primers and PCR conditions used to amplify these sites.

W O 2014/144592 ״ PCT/US2014/029068 WO 2014/144592 PCT/US2014/029068 Determination of RGN-induced on- and off-target mutation frequencies in human cells ForU2OS.EGFP and K562 cells, 2 x 105 cells were transfected with 250 ng of gRNA expression plasmid or an empty U6 promoter plasmid (for negative controls), 750 ng of Cas9 expression plasmid, and 30 ng of td-Tomato expression plasmid using the 4D Nucleofector System according to the manufacturer ’s instructions (Lonza). For HEK293 cells, 1.65 x 105 cells were transfected with 125 ng of gRNA expression plasmid or an empty U6 promoter plasmid (for the negative control), 375 ng of Casexpression plasmid, and 30 ng of a td-Tomato expression plasmid using Lipofectamine LTX reagent according to the manufacturer ’s instructions (Life Technologies). Genomic DNA was harvested from transfected U2OS.EGFP, HEK293, or K562 cells using the QIAamp DNA Blood Mini Kit (QIAGEN), according to the manufacturer ’s instructions. To generate enough genomic DNA to amplify the off-target candidate sites, DNA from three Nucleofections (for U2OS.EGFP cells), two Nucleofections (for K562 cells), or two Lipofectamine LTX transfections was pooled together before performing T7EI. This was done twice for each condition tested, thereby generating duplicate pools of genomic DNA representing a total of four or six individual transfections. PCR was then performed using these genomic DNAs as templates as described above and purified using Ampure XP beads (Agencourt) according to the manufacturer ’s instiuctions. T7EI assays were performed as previously described (Reyon et al., 2012, supra). DNA sequencing of NHEJ-mediated indel mutations Purified PCR products used for the T7EI assay were cloned into Zero Blunt TOPO vector (Life Technologies) and plasmid DNAs were isolated using an alkaline lysis miniprep method by the MGH DNA Automation Core. Plasmids were sequenced using an M13 forward primer (5’ - GTAAAACGACGGCCAG -3’ (SEQ ID NO: 1059) by the Sanger method (MGH DNA Sequencing Core).

Example la. Single Nucleotide Mismatches To begin to define the specificity determinants of RGNs in human cells, a large-scale test was performed to assess the effects of systematically mismatching various positions within multiple gRNA/target DNA interfaces. To do this, a quantitative human cell-based enhanced green fluorescent protein (EGFP) disruption WO 2014/144592 PCT/US2014/029068 assay previously described (see Methods above and Reyon ct al., 2012, supra) that enables rapid quantitation of targeted nuclease activities (Fig. 2B)was used. In this assay, the activities of nucleases targeted to a single integrated EGFP reporter gene can be quantified by assessing loss of fluorescence signal in human U2OS.EGFP cells caused by inactivating frameshift insertion/deletion (indel) mutations introduced by error prone non-homologous end-joining (NHEJ) repair of nuclease-induced double- stranded breaks (DSBs) (Fig. 2B).For the studies described here, three ~100nt single gRNAs targeted to different sequences within EGFP were used, as follows: EGFP Site 1 GGGCACGGGCAGCTTGCCGGTGG (SEQ ID NO:9) EGFP Site 2 GATGCCGTTCTTCTGCTTGTCGG (SEQ ID NO:10) EGFP Site 3 GGTGGTGCAGATGAACTTCAGGG (SEQ ID NO: 11) Each of these gRNAs can efficiently direct Cas9-mediated disruption of EGFP expression (see Example le and 2a,and FIGs. 3E (top) and 3F (top)). In initial experiments, the effects of single nucleotide mismatches at 19 of nucleotides in the complementary targeting region of three EGPP-targeted gRNAs were tested. To do this, variant gRNAs were generated for each of the three target sites harboring Watson-Crick transversion mismatches at positions 1 through (numbered 1 to 20 in the 3 ’ to 5 ’direction; see Fig. 1)and the abilities of these various gRNAs to direct Cas9 ־mediated EGFP disruption in human cells tested (variant gRNAs bearing a substitution at position 20 were not generated because this nucleotide is part of the U6 promoter sequence and therefore must remain a guanine to avoid affecting expression.)For EGFP target site #2, single mismatches in positions 1 - 10 of the gRNA have dramatic effects on associated Cas9 activity (Fig. 2C,middle panel), consistent with previous studies that suggest mismatches at the 5’ end of gRNAs are better tolerated than those at the 3’ end (Jiang et al., Nat Biotechnol 31, 233-239 (2013); Cong et al., Science 339, 819-823 (2013); Jinek et al., Science 337, 816-821 (2012)). However, with EGFP target sites #1 and #3, single mismatches at all but a few positions in the gRNA appear to be well tolerated, even within the 3 ’ end of the sequence. Furthermore, the specific positions that were sensitive to mismatch differed for these two targets (Fig. 2C,compare top and bottom panels) - for PCT/US2014/029068 WO 2014/144592 example, target site #1 was particularly sensitive to a mismatch at position 2 whereas target site #3 was most sensitive to mismatches at positions 1 and 8.

Example lb. Multiple Mismatches To test the effects of more than one mismatch at the gRNA/DNA interface, a series of variant gRNAs bearing double Watson-Crick transversion mismatches in adjacent and separated positions were created and the abilities of these gRNAs to direct Cas9 nuclease activity were tested in human cells using the EGFP disruption assay. All three target sites generally showed greater sensitivity to double alterations in which one or both mismatches occur within the 3’ half of the gRNA targeting region. However, the magnitude of these effects exhibited site-specific variation, with target site #2 showing the greatest sensitivity to these double mismatches and target site #1 generally showing the least. To test the number of adjacent mismatches that can be tolerated, variant gRNAs were constructed bearing increasing numbers of mismatched positions ranging from positions 19 to 15 in the 5’ end of the gRNA targeting region (where single and double mismatches appeared to be better tolerated).Testing of these increasingly mismatched gRNAs revealed that for all three target sites, the introduction of three or more adjacent mismatches results in significant loss of RGN activity. A sudden drop off in activity occurred for three different EGFP-targeted gRNAs as one makes progressive mismatches starting from position 19 in the 5’ end and adding more mismatches moving toward the 3’ end. Specifically, gRNAs containing mismatches at positions 19 and 19+18 show essentially full activity whereas those with mismatches at positions 19+18+17, 19+18+17+16, and 19+18+17+16+15 show essentially no difference relative to a negative control (Figure 2F). (Note that we did not mismatch position 20 in these variant gRNAs because this position needs to remain as a G because it is part of the U6 promoter that drives expression of the gRNA.)Additional proof of that shortening gRNA complementarity might lead to RGNs with greater specificities was obtained in the following experiment: for four different EGFP-targeted gRNAs (Figure 2H),introduction of a double mismatch at positions 18 and 19 did not significantly impact activity. However, introduction of another double mismatch at positions 10 and 11 then into these gRNAs results in near complete loss of activity. Interestingly introduction of only the 10/11 double mismatches does not generally have as great an impact on activity.

PCT/US2014/029068 WO 2014/144592 Taken together, these results in human cells confirm that the activities of RGNs can be more sensitive to mismatches in the 3 ’ half of the gRNA targeting sequence. However, the data also clearly reveal that the specificity of RGNs is complex and target site-dependent, with single and double mismatches often well tolerated even when one or more mismatches occur in the 3’ half of the gRNA targeting region. Furthermore, these data also suggest that not all mismatches in the ’ half of the gRNA/DNA interface are necessarily well tolerated.In addition, these results strongly suggest that gRNAs bearing shorter regions of complementarity (specifically -17 nts) will be more specific in their activities. We note that 17 nts of specificity combined with the 2 nts of specificity conferred by the PAM sequence results in specification of a 19 bp sequence, one of sufficient length to be unique in large complex genomes such as those found in human cells.

Example to. Off-Target Mutations To determine whether off-target mutations for RGNs targeted to endogenous human genes could be identified, six single gRNAs that target three different sites in the VEGFA gene, one in the EMX1 gene, one in the RNF2 gene, and one in the FANCF gene were used (Table 1and Table A).These six gRNAs efficiently directed Cas9-mediated indels at their respective endogenous loci in human U2OS.EGFPcells as detected by T7 Endonuclease I (T7EI)assay (Methodsabove and Table1). For each of these six RGNs, we then examined dozens of potential off- target sites (ranging in number from 46 to as many as 64) for evidence of nuclease- induced NHEJ-mediated indel mutations in U2OS.EGFP cells. The loci assessed included all genomic sites that differ by one or two nucleotides as well as subsets of genomic sites that differ by three to six nucleotides and with a bias toward those that had one or more of these mismatches in the 5 ’ half of the gRNA targeting sequence (Table B).Using the T7EI assay, four off-target sites (out of 53 candidate sites examined) for VEGFA site !,twelve (outof 46 examined) for VEGFA site 2, seven (out of 64 examined) for VEGFA site 3 and one (out of 46 examined) for the EMXsite (Table 1and Table B)were readily identified. No off-target mutations were detected among the 43 and 50 potential sites examined for the RNF2 or FANCF genes, respectively (Table B).The rates of mutation at verified off-target sites were very high, ranging from 5.6% to 125% (mean of 40%) of the rate observed at the intended target site (Table 1).These bona fide off-targets included sequences with PCT/US2014/029068 WO 2014/144592 mismatches in the 3’ end of the target site and with as many as a total of five mismatches, with most off-target sites occurring within protein coding genes (Table 1).DNA sequencing of a subset of off-target sites provided additional molecular confirmation that indel mutations occur at the expected RGNcleavage site (Figs. 8A- C).

WO 2014/144592 PCT/US2014/029068 Table 1 On- and off-target mutations induced by RGNs designed to endogenous human genes TargetSite nameSequenceSEQ ID NO:Indel Mutation Frequency (%) ± SEMGene U20S.EGFP HEK293 K562 Target ^VEGFA Site 1) T1 GGGIGGGGGGAGTTTGCTCCTGG 1059 2 6.0 ± 2.9 10.5 + 0.07 3.33 + 0.42 VEGFAOTl -3 GGATGGAGGGAGTTTGCTCCTGG 1060 25.7 ± 9.1 18.9 ± 0.77 2.93 ± 0.04 TGDCG3OT1-4 GGGAGGGTGGAGTTTGCTCCTGG 1061 9.2 ± 0.8 8.32 ± 0.51 N.D. LOC116'S37OT1-6 CGGGGGAGGGAGTTTGCTCCTGG 1062 5.3 + 0.2 3.67 + 0.09 N. D. CACNA2DOT1-11 GGGGAGGGGAAG TTTGCTCCTGG 1063 17.1 + 4.7 8.54 + 0.16 N.D.

Target (VEGFA Site 2) T2 GACCCCCTCCACCCCGCCTCCGG 1064 50.2 ± 4.9 38.6 ± 1.92 15.0 ± 0.25 VEGFAOT2-1 GACCCCCCCCACCCCGCCCCCGG 1065 14.4 ± 3.4 33.6 ± 1.17 4.10 + 0.05 FMN1OT2-2 GGGCCCCTCCACCCCGCCTCTGG 1066 20.0 ± 6.2 15.6 ± 0.30 3.00 ± 0.06 PAX6OT2-6 CTACCCCTCCACCCCGCCTCCGG 1067 8.2 + 1.4 15.0 + 0.64 5.24 ± 0.22 PAPD7OT2-9 GCCCCCACCCACCCCGCCTCTGG 1068 50.7 ± 5.6 30.7 ± 1.44 7.05 + 0.48 LAMA3012-15 TACCCCCCACACCCCGCCTCTGG 1069 9.7 + 4.5 6.97 + 0.10 1.34 ± 0.15 SPNS3or2-17 ACACCCCCCCACCCCGCCTCAGG 1070 14.0 12.8 12.3 1 0.45 1.80 + 0.03OT2-19 ATTCCCCCCCACCCCGCCTCAGG 1071 17.0 + 3.3 19.4 ± 1.35 N. n. HDLBPOT2-20 CCCCACCCCCACCCCGCCTCAGG 1072 6.1 1 1.3 N.D. N.D. ABLIM1OT2-23 CGCCCTCCCCACCCCGCCTCCGG 1073 44.4 + 6.7 28.7 ± 1.15 4.18 ± 0.37 CALYOT2-24 CTCCCCACCCACCCCGCCTCAGG 1074 62.8 1 5.0 29.8 ± 1.08 21.1 + 1.68OT2-29 TGCCCCTCCCACCCCGCCTCTGG 1075 13.8 ± 5.2 N.D. N.D. AC DYOT2-34 AGGCCCCCACACCCCGCCTCAGG 1076 2.8 ± 1.5 N.D. N.D.

Target (VEGFA Site 3) T3 GGTGAGTGAGTGTGTGCGTGTGG 1077 4 9.4 + 3.8 35.7 + 1.26 27.9 + 0.52 VEGFAOT3-1 GGTGAGTGAGTGTGTGTGTGAGG 1078 7.4 ± 3.4 8.97 + 0.80 N.D. (abParts)OT3-2 AGTGAGTGAGTGTGTGTGTGGGG 1079 24.3 ± 9.2 23.9 + 0.08 8.9 ± 0.16 MAXOT3-4 GCTGAGTGAGTGTATGCGTGTGG 108020.9 111.811.2 + 0.23 N.D.OT3-9 GGTGAG’TGAGTGCGTGCGGGTGG 1081 3.2 + 0.3 2.34 + 0.21 N.D. TPCN2OT3-17 GTPTGAGTGAATGTGTGCGTGAGG 1082 2.9 ± 0,2 1.27 ± 0.02 N.D. SLIT1OT3-18 TGTGGGTGAGTGTGTGCGTGAGG 1083 13.4 ± 4.2 12.1 + 0.24 2.42 ± 0.07 COMDAOT3-20 AGAGAG T GA G '1' G '1' G T G CAT GA G G 1084 16.7 ± 3.5 7.64 + 0.05 1.18 1 0.01Target (EMX1)T4 GAGTCCGAGCAGAAGAAGAAGGG 1085 42.1 ± 0.4 2 6.0 + 0.70, 10.7 ± 0.50 EMX1OT4-1 GAGTTAGAGCAGAAGAAGAAAGG 1086 16.8 + 0.2 8.43 ± 1.32 2.54 + 0.02 HCN1Target 5 (ENF2) T5 G’TCATCTTAGTCATTACCTGTGG 1087 6.0 ׳ ± 6.6 2— —RNF2Target 6 (FA^CF) T6 GGAATCCC’TTCTGCAGCACCAGG 1088 33.2 ±6.5— —FANCF"OT" indicates off-target sites (with numbering of sites as in Table E).Mismatches from the on-target (within the 20 bp region to which the gRNA hybridizes) are highlighted as bold, underlined text. Mean indel mutation frequencies in U2OS.EGFP, HEK293, and K562 cells were determined as described in Methods.Genes in which sites were located (if any) are shown. All sites listed failed to show any evidence of modification in cells transfected with Cas9 expression plasmid and a control U6 promoter plasmid that did not express a functional gRNA. N.D. = none detected; — = not tested.

PCT/US2014/029068 WO 2014/144592 Example Id. Off-Target Mutations in Other Cell Types Having established that RGNs can induce off-target mutations with high frequencies in U2OS.EGFP cells, we next sought to determine whether these nucleases would also have these effects in other types of human cells. We had chosen U2OS.EGFP cells for our initial experiments because we previously used these cells to evaluate the activities of TALENs15 but human HEK293 and K562 cells have been more widely used to test the activities of targeted nucleases. Therefore, we also assessed the activities of the four RGNs targeted to VEGFA sites 1,2, and 3 and the EMXI site in HEK293 and K562 cells. We found that each of these four RGNs efficiently induced NHEJ-mediated indel mutations at their intended on-target site in these two additional human cell lines (as assessed by T7EI assay) (Table 1),albeit with somewhat lower mutation frequencies than those observed in U20S.EGFP cells. Assessment of the 24 off-target sites for these four RGNs originally identified in U2OS.EGFP cells revealed that many were again mutated in HEK293 and K562 cells with frequencies similar to those at their corresponding on-target site (Table 1).As expected, DNA sequencing of a subset of these off-target sites from HEK293 cells provided additional molecular evidence that alterations are occurring at the expected genomic loci (Figs. 9A-C).We do not know for certain why in HEK293 cells four and in K562 cells eleven of the off-target sites identified in U2OS.EGFP cells did not show detectable mutations. However, we note that many of these off-target sites also showed relatively lower mutation frequencies in U2OS.EGFP cells. Therefore, we speculate that mutation rates of these sites in HEK293 and K562 cells may be falling below the reliable detection limit of our T7EI assay (~2-5%) because RGNs generally appear to have lower activities in HEK293 and K562 cells compared with U2OS.EGFPcells in our experiments. Taken together, our results in HEK293 and K562 cells provide evidence that the high-frequency off-target mutations we observe with RGNs will be a general phenomenon seen in multiple human cell types.

Example le. Titration of gRNA- and Cas9-expressing plasmid amounts used for the EGFP disruption assay Single gRNAs were generated for three different sequences (EGFP SITES 1-3, shown above) located upstream of EGFP nucleotide 502, a position at which the introduction of frameshift mutations via non-homologous end-joining can robustly WO 2014/144592 PCT/US2014/029068 disrupt expression of EGFP (Maeder, M.L. ct al., Mol Cell 31,294-301 (2008); Reyon, D. et al., Nat Biotech 30, 460-465 (2012)).For each of the three target sites, a range of gRNA-expressing plasmid amounts (12.5 to 250 ng) was initially transfected together with 750 ng of a plasmid expressing a codon-optimized version of the Cas9 nuclease into our U2OS .EGFP reporter cells bearing a single copy, constitutively expressed EGFP-PEST reporter gene. All three RGNsefficiently disrupted EGFPexpression at the highest concentration of gRNA-cncoding plasmid (250 ng) (Fig. 3E (top)).However, RGNs for target sites #1 and #3 exhibited equivalent levels of disruption when lower amounts of gRNA-expressing plasmid were transfected whereas RGN activity at target site #2 dropped immediately when the amount of gRNA-expressing plasmid transfected was decreased (Fig. 3E(top)). The amount of Cas9-encoding plasmid (range from 50 ng to 750 ng) transfected into our U2OS.EGFP reporter cells was titrated and EGFP disruption assayed. As shown in Fig. 3F (top),target site #1 tolerated a three-fold decrease in the amount of Cas9-encoding plasmid transfected without substantial loss of EGFP disruption activity. However, the activities of RGNs targeting target sites #2 and #decreased immediately with a three-fold reduction in the amount of Cas9 plasmid transfected (Fig. 3F (top)).Based on these results, 25ng/250ng, 250ng/750ng, and 200ng/750ng of gRNA-/Cas9-expressing plasmids were used for EGFP target sites #1, #2, and #3, respectively, for the experiments described in Examples la-Id.The reasons why some gRNA/Cas9 combinations work better than others in disrupting EGFP expression is not understood, nor is why some of these combinations are more or less sensitive to the amount of plasmids used for transfection. Although it is possible that the range of off-target sites present in the genome for these three gRNAs might influence each of their activities, no differences were seen in the numbers of genomic sites that differ by one to six bps for each of these particular target sites (Table C)that would account for the differential behavior of the three gRNAs.

PCT/US2014/029068 WO 2014/144592 Table C Numbers of off-target sites in the human genome for six RGNs targeted to endogenous human genes and three RGNs targeted to the EGFP reporter gene Target Site Number of mismatches to on-target site 0 1 ? 3 4 5 6 Target Site 1} 1 1 4 32 280 2175 13873 Target 2 (1/fGF/l Site 2) 1 0 2 35 443 3889 17398 Target 3 (i/£GFA Site 3) 1 1 17 377 6028 13398 35517 Target 4 (£MX) 1 0 1 18 276 2309 15731 Targets (/?/VF2) 1 0 0 6 116 976 7443 Target 6 (FZUVCF) 1 0 1 18 271 1467 9551 EGFP Target Site #1 0 0 3 10 156 1365 9755 EGFP Target Site #2 0 0 0 11 96 974 7353 EGFP Target Site #3 0 0 1 14 165 1439 10361 Off-target sites for each of the six RGNs targeted to the VEGFA, RNF2, FANCF, and EMX1 genes and the three RGNs targeted to EGFP Target Sites #1, #and #3 were identified in human genome sequence build GRCh37. Mismatches were only allowed for the 20 nt region to which the gRNA anneals and not to the PAM sequence.

Example 2: Shortening gRNA complementarity length to improve RGN cleavage specificity It was hypothesized that off-target effects of RGNs might be minimized without compromising on-target activity simply by d ecreasing the length of the gRNA-DNA interface, an approach that at first might seem counterintuitive. Longer gRNAs can actually function less efficiently at the on-target site (see below and Hwang et al., 2013a; Ran et al., 2013). In contrast, as shown above in Example 1, gRNAs bearing multiple mismatches at their 5’ ends could still induce robust cleavage of their target sites (Figures 2A and 2C-2F), suggesting that these nucleotides might not be required for full on-target activity. Therefore, it was hypothesized that truncated gRNAs lacking these 5 ’ nucleotides might show activities comparable to full-length gRNAs (Figure 2A). It was speculated that if the 5 ’ nucleotides of full-length gRNAs are not needed for on-target activity then their presence might also compensate for mismatches at other positions along the gRNA- target DNA interface. If this were true, it was hypothesized that gRNAs might have greater sensitivity to mismatches- and thus might also induce substantially lower levels of Cas9-mediated off-target mutations (Figure 2A).

PCT/US2014/029068 WO 2014/144592 Experimental Procedures The following experimental procedures were used in Example 2. Plasmid construction All gRNA expression plasmids were assembled by designing, synthesizing,annealing, and cloning pairs of oligonucleotides (IDT) harboring the complementarity region into plasmid pMLM3636 (available from Addgene) as described above (Example 1). The resulting gRNA expression vectors encode a ~100 nt gRNA whose expression is driven by a human U6 promoter. The sequences of all oligonucleotides used to construct gRNA expression vectors are shown in T able D.The Cas9 D1 0Anickase expression plasmid (pJDS271) bearing a mutation in the RuvC endonuclease domain was generated by mutating plasmid pJDS246 using a QuikChange kit (Agilent Technologies) with the following primers: Cas9 D10A sense primer 5’- tggataaaaagtattctattggtttagccateggcactaattccg-3 ‘ (SEQ ID NO:1089); Cas9 D10A antisense primer 5’-cggaattagtgccgatggctaaaccaatagaatactttttatcca3 ־’ (SEQ IDNO: 1090). All the targeted gRNA plasmids and the Cas9 nickase plasmids used in this study are available through the non-profit plasmid distribution service Addgene (addgene.org/crispr-cas ).

Table D Sequences of oligonucleotides used to construct gRNA expression plasmids EGFP Target Site 1 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 3') SEQ ID HO: oligonucleotide 2 (5' to 3') SEQ ID עס:؛£؛؛«•|^|[gw|B lit ®IBlACACCGCACGGGCAGCTTGCCGGG 1091 AAAACGGGGCAAGCTGCCCGTGCG 1130 .G C A C G V G c A G C T r G C G ACACCGCACGGGCAGCTTGCCGCG 1092 AAAACGCGGCAAGCTGCCCGTGCG 1191.G XX A C G G G c A G c T T G rrG ACACCGCACGGGCAGCTTGCCCGG 1093 AAAACCGGGCAAGCZGCCCGTGCG 1192 .Go A c G G G c A G c T 1'׳ W('ACACCGCACGGGCAGCTTGCGGGG1094 AANACCCCGCAAGCTGCCCGTGCG 1183.G c A c G G G c A G c T Tr:C G G ACACCGCACGGGCAGCTTGGCGGG 1095 AAAACCCGCCAAGCTGCCCGTGCG 1194 .G c A c G G G c A G c­ ז ­T c L G G ACACCGCACGGGCAGCTTGCCGGG 1096 AAAACCCGGGAAGCTGCCCGT GCG 1185-G c A cG G G c A G c ! m )؛ C c G G ACACCGCACGGGCAGCTAGCCGGG 1097 AAAACCCGGCTAGCTGCCCGTGCG 1186.G c A c G G G c A G 1: r G C c G G ACACCGCACGGGCAGCATGCCGGG 1098 AAAACCCGGCATGCTGCCCGTGCG 1187 .G c A c G G c A Gill IVr G C c G Sa ACACCGCACGGGCAGGTTGCCGGG 1099 AAAACCCGGCAACCTGCCCGTGCG 1188 .G a A c G G<' A c T r G C c G G ACACCGCACGGGCAGCTTGCCGGG 1100 AAAACCCGGCAAGGTGCCCGTGCG 1199.G c A c G G c G c I r G c c G G ACACCGCACGGGCTGCTTGCCGGG 1101 AAAACCCGGCAAGCAGCCCGTGCG 1190.Go A c GnG #g A G c T 1 G c c G G ACACCGCACGGGGAGCTTGCCGGG 1102 AAAACCCGGCAAGCTCCCCGTGCG 1191.G c A c G G I ■ A G c T 1 G c/־IG G ACACCGCACGGCCAGCTTGCCGGG 1103 AAAACCCGGCAAGCTGGCCGTGCG 1192.G A c (j A G c T T G c c G G ACACCGCACGCCCAGCTTGCCGGG 1104 AAAACCCGGCAAGCTGCGCGTGCG 1193.G c A c G A GcT T G c c G G ACACCGCACGGGCAGCTTGCCGGG 1105 AAAACCCGGCAAGCTGCCGGTGCG 1194 .Gr. A U G c A G c T T G c c G G ACACCGCAGGGC-CAGCTTGCCGGG 1106 AAAACCCGGCAAGCTGCCCCTGCG 1195 .Gwn rG G G c A G כ T T G c c G G ACACCGCTCGGGCAGCTTGCCGGG 1107 AAAACCCGGCAAGCTGCCCGAGCG 1196.G A G G G c A G c T r G c c G G ACACCGGACGGGCAGCTTGCCGGG 1108 AAAACCCGGCAAGCTGCCCSTCCG 1197 .G c A c G G G a A G c T r c; ^:1 ACACCGCACGGGCAGCTTGCCGCG 1109 AAAACGGGGCAAGCTGCCCGTGCG 1198 .G c A c G |J G c A G c T r ؛ C■(a G ACACCGCACGGGCAGCTTGGGGGG 1110 AAAACCCCCCAAGCTGCCCGTGCG 1199.G c A c G G G c A G c T /.•u .. :O:c G G ACACCGCACGGGCAGCTAGCCGGG 1111 AAAACCCGGGTAGCTGCCCGTGCG 120G.G c A c G kJ G c A G 1; c G G ACACCGCACGGGCAGGATGCCGGG 1112 AAAACCCGGCATGCTGCCCGTGCG 1201.

W O 2014/144592 PCT/US2014/029068 Table D Sequences of oligonucleotides used to construct gRNA expression plasmids — G c A c G G G (' M* c T rGc C G G ACACCGCACGGGCTCCTTGCCGGG 1113 AAAACCCGGCAAGGAGCCCGTGCG 1202. —Gr A r־ G G ؛،؛؛، A G c T r G c C G G ACACCGCACGGCGAGCTTGCCGGG 1114 AAAACCCGGCAAGCTCGCCGTGCG 1203. —G A•- G 1" A G c T r G c C G G ACACCGCACCCGCAGCTTGCCGGG 1115 AAAACCCGGCAAGCTGCGGGTGCG 1204. :G $8 G G G c A G c T r G c c G G ACACCGCTGGGGCAGCTTGCCGGG 1116 AAAACCCGGCAAGCTGCCCCAGCG־ . 1205G #1 HiG G G c A G c T r G c c G G A.CACCGGTCGGGCAGCTTGCCGGG 1117 AAAACCCGGCAAGCTGCCCGACCG1206. ~ ECFP Target Sita 2 IS 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5’ to 3') oligonucleotide 2 (5' to 3')404 ן#־I'i'i ؛€؛؛،؛ S ؛ i#iwo! HIorHlV : ACACCGCCGTTCTTCTGCTT’GTG 1118 AAAACACAAGCAGAAGAACGGCG 1207.(•G T T c T T c T 1' r G sSg ACACCGCCGTTCTTCTGCTTGAG 1119 AAAACTCAAGCAGAAGAACGGCG 1208 .G c c G T T c T T c T G c T r W T ACACCGCCGTTCTTCTGCTTCTG 1120 AAAACAGAA.GCAGAAGAACGGCG 1209 .G c c G T T c T r 0 T G TםG T ACACCGCCGTTCTTCTGCTAGTG 1121 AAAACACTAGCAGAAGAACGGCG 1210.G c c G T T c r r c T G c r G T ACACCGCCGTTCTTCTGCATGTG 1122 AAAACACATGCAGAAGAACGGCG 1211.G c c G T T c t r c T G T T G T ACACCGCCGTTCTTCTGGTTGTG 1123 AAAACACAAGCAGAAGAACGGCG 1212.G c c GTT c r r c T c ■ r G 1' ACACCGCCGTTCTTCTCCTTGTG 1124 AAAACACAAGGAGAAGAACGGCG 1213.G c c GוקT c t T r#G c T r G T ACACCGCCGTTCTTCAGCTTGTG 1125 AAAACACAAGCTGiAAGAACGGCG 1214 .G c c G r T c r V $ T G c T T G T ACACCGCCGTTCTTGTGCTTGTG 1126 AAAACACAAGCACAAGAACGGCG 1215-u c c G r T c T 0 '1' G c T TGT ACACCGCCGTTCTACTGCTTGTG 1127 AAAACACAA-GCAGTAGAACGGCG 1216.G nc G r T c r (2 T G c T T G T ACACCGCCGTTCATCTGCPTGTG 1128 AAAACACM£CAGATGAACGGCG 1217.c c G r THUT T c T G c T T G T ACACCGCCGTTGITCTGCTTGTG 1129 AAAACACAAGCAGAAGAACGGCG 1219 .G c c Tf T r c T G c T T G T ACACCGCCGTACT TCTGCTTGTG 1130 AAAACACAA.GCAGAAGTACGGCG 1219.G c c G T c T T c T G c T T G T ACACCGCCGATCTTCTGCTTGTG 1131 AAAACACAAGCAGAAGATCGGCG 1220 .G c c k T c T T c T G c T T G T ACACCGCCCTTCTTCTGCTTGTG 1132 AAAACACAAGCAGAAGAZ1GGGCG 1221.G eI®״. 1 T c T r ס T G c T T G T ACACCGCGGTT C TTCTGCTTGTG 1133 AAAACACAAGCAGAAGAACGGCG 1222 .G Tc T T c T G c T T G T ACACCGGCGTTCTTCTGCTTGTG 113-1 AAAACACAAGCAGAAGAACGGCG 1223 .G c c T T c T T c T G c T r Bg&i ACACCGCCGTTCTTCTGCTTGAG 1135 AAAACTG71AGCAGMGAACGGCG 1224 .

W O 2014/144592 PCT/US2014/029068 Table D Sequences of oligonucleotides used to construct gRNA expression plasmids G C C G r T CnT ׳- 1.I1 G׳ 1G T ACACCGCCGTTCTTCTGCAAGTG 1136 AAAACACTTGCAGAAGAACGGCG 1225.G C C G T T Cri׳■■■T א T T G r ACACCGCCGTTCTTCTCGTTGTG 1137 AAAACACAACGAGAAGAACGGCG 1226.G c C G r T C I (j L T ؟ G T A.CACCGCCGTTCTTGAGCTTGTG 1138 AAAACACAAGCTCEAGAACGGCG . ־ 122G 0 C G 1' T C 5^ T Ij c I T G T ACACCGCCGTTCAACTGCTTGTG 1139 AAAACACAAGC AG T T GAAC GGC G 1226.G c C G 1'OraT T c T G c T T G T ACACCGCCGTAGTTCTGCTTGTG 1140 AAAACACAAGCAGAACTACGGCG 1229.G c C T CTT c T G c T T 1J T ACACCGCCCATCTTCTGCTTGTG 1141 AAAACACAAGCAGAAGATGGGCG 1230.G nm ־ G r r C T TT G c T T T ACACCGGGGTTCTTCTGCTTGTG 1142 AAAACACAAGCAGAAGAACCCCG 1231.EGFP Target Site 3IS 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 31) oligonucleotide 2 (S' to 3')QI،؛ :HIHI IIIw.MTi® tesמACACCGGTGCAGATGAACTTCAG 1143 AAAACTCTAGTTCATCTGCACCG 1232 .G C r G C A G A T A Ar_•..IIIACACCGGTGCAGATGAACTTCTG 1144 AAAACTCAAGTTCATCTGCACCG 1233.G G r G c A G A T G A A C T TiiM/ ACACCGGTGCAGATGAACTTCAG 1145 AAAACTGTAGTTCAT’CTGCACCG 1234 .G G T G c A G A G A A 1'. T A A.CACCGGTGCAGATGAACTACAG 1146 AAAACTGATGTTCATCTGCACCG 1235.G G r G c A G AmG A A C T c A ACACCGGTGCAGATGAACATCAG 1147 AAAACTGAACTTCATCTGCACCG 1236.GT G c A G A i. G A A '1' ,C c A ACACCGGTGCAGATGAACTTCAG 1148 AAAACTGAAGATCATCTGCACCG 1237 .G G r G c A G A W O 2014/144592 PCT/US2014/029068 th Table D Sequences of oligonucleotides used to construct gRNA expression plasmids G T G c A G A I G A A C T T A ACACCGCTGCAGATGAPCTTCAG 1159 AAAACTGAAGTTCATCTGCAGCG 1248. G G T G c A G A T G A A c : 1 ׳ '1' Bl0 ACACCGGTGCAGATGAACTTGTG 1160 AAAACACAAGTTCATCTGCACCG 1249 .G G T G c A G A T G A A c M • A ACACCGGTGCAGA’TGAACAACAG 1161 AAAACTGTTGTTCATCTGCACCG 1250.G G T G c A G A T 1:1 A Sts-in T T;י A ACACCGGTGCAGATGATGTTCAG 1162 AAAACTGAACATCATCTGCACCG 1251. G G T G c A G A Ta_ T T C A ACACCGGTGCAGATCTACTTCAG 1163 AAAACTGAAGTAGATCTGCACCG 1252.G G T G c A G SB A A c I T C A ACACCGGTGCAGTAGAACTTCAG 1164 AAAACTGAAGTTCTACTGCACCG 1253.G G 1r G c :®g A 1' A A c T T c A ACACCGGTGCTCATGAACTTCAG 1165 AAAACTGAAGTTCATGAGCACCG 1254 .G (i TA A T G A A c T T c A ACACCGGTCGAGAIGA?.C1rCAG 1166 AAAACTGAAGTTCATCTCGACCG 1255 . G G c A G A T G A A c T T c A ACACCGCAGCAGATGAACTTCAG 1167 AAAACTGAAGTTCATCTGCTGCG 1256. Endogenous Target 1 tVEGEA Site 1 tru-gRNA) : 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (S' to 3') oligonucleotide 2 (5' to 3') G T G G G G G G A G T T T G C T C C ACACCGTGGGGGGAGTTTGCTCCG 1168 AAAACGGAGCAAACTCCCCCCACG 1257 . Endogenous Target 3 (VEGFA Site 3 tru-gRNA): 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5׳ to 3•) oligonucleotide 2 (5' to 3') G A G T G A G T G T G T G C G T G ACACCGAGTGAGTGTGTGCGTGG 1169 AAAACCACGCACACACTCACTCG 1258. > o Endogenous Target 4 (EWU site 1 tru-gRNA) : 5 20 19 18 17 16 15 14 13 12 11 10 9 8 ד 6 5 4 3 2 1 oligonucleotide 1 (S' to 3") oligonucleotide 2 (5■ to 3') ס o a T C C a A C C A C A A C A A C A A ACACCGTCCGAGCAGAAGAAGAAG 1170 AAAACTTCTTCTTCTGCTCGGACG ket CM 1 —1 CTLA full-Length gRNA ؛ 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 3') oligonucleotide 2 (S' to 3’) 786- G C A G A T G T A G T G T T T C C A C A ACACCGCAGATGTAGTGTTTCCACAG 1171 AAJkACTGGGGAAACACTACArCTGCG 1260. § CTLA tru-gRNA § 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 3') oligonucleotide 2 (5' to 3') G A T G T A G T G T T T C C A C A ACACCGATGTAGTGTTTCCACAG 1172 AAAACTGTGGAAACACTACATCG 1261. 8 0 2014/144592 PCT/US2014/0 Table D Sequences of oligonucleotides used to construct gRNA expression plasmids VECFA site 4 full-length gRNA 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5׳ to 3') oligonucleotide 2 (5׳ to 3״) I C C C T C T T T A G C C A G A G C a G ACACCTCCCTCIT’TAGCCAGAGCCGG 1173 AAAACCGGCICTGGCTAAAGAGGGAG 1262 . EMX1 site 2 full-length gRNA 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 3') oligonucleotide 2 (5' to 3') G C C G T T T G T A c T T I G T C C r C ACACCGCCGTTTGTACTTTGTCCTCG 1174 AAAACGAGGACAAAGTACAAACGGCG 1263 . EMX1 site 2 tzu-gRNA 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 3") oligonucleotide 2 (5' to 3') G T I T G T A c r T I G T C c T C ACACCGTTTGTACTTTGTCCTCG 1L75 AAAACGAGGACAAAGTACAAACG 1264 . EMX1 site 3 full-length gRNA. 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5r ־to 3r) oligonucleotide 2 (51 to 3') G G G A A G A C T G A G G C I A C A r A ACACCGGGAAGACTGAGGCTACATAG 1176 AAAACTATGTAGCCTCAGTCTTCCCG 1265 . EMX1 site 3 tzu—gRNA 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 3') oligonucleotide 2 (5' to 3') G A A G A C T G A G G C T A C A T A ACACCGAAGAC T GAGGCTACATAG 1177 AAAACTATGTAGCCTCAGTCTTCG 1266. g EMX1 site 4 full-length gRNA 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 3') oligonucleotide 2 (S' to 3') pOQ G A G G C C C C C A G A G C A G C C A C ACACCGAGGCCCCCAGAGCAGCCACG 1178 AAAACGTGGCTGCTCTGGGGGCCTCG CD 1267. * EHX1 site 4 tru-gRNA g 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 oligonucleotide 1 (5' to 3' ) oligonucleotide 2 (S' to 3') '86-C G C C C C C A G A G C A G C C A a ACACCGCCCCCAGAGCAGCCACG 1179 AAAACGTGGCTGCTCTGGGGGCG ....... cu 1266. g W O 2014/144592 PC T/U S2014/029068 WO 2014/144592 PCT/US2014/029068 Human cell-based EGFP disruption assay U2OS.EGFP cells harboring a single-copy, integrated EGFP-PEST gene reporter have been previously described (Reyon et al., 2012). These cells were maintained in Advanced DMEM (Life Technologies) supplemented with 10% FBS, mM GlutaMax (Life Technologies), penicillin/streptomycin and 400 pg/ml G418. To assay for disruption of EGFP expression, 2 x 105 U2OS.EGFP cells were transfected in duplicate with gRNA expression plasmid or an empty U6 promoter plasmid as a negative control, Cas9 expression plasmid (pJDS246) (Example 1 andFu ct al., 2013), and 10 ng of td-Tomato expression plasmid (to control for transfection efficiency) using a LONZA 4D-Nucleofector™, with SE solution and DN1program according to the manufacturer ’s instructions. We used 25 ng/250 ng, 2ng/750 ng, 200 ng/750 ng, and 250 ng/750 ng of gRNA expression plasmid/Casexpression plasmid for experiments with EGFP site #1, #2, #3, and #4, respectively. Two days following transfection, cells were trypsinized and resuspended in Dulbecco ’s modified Eagle medium (DMEM, Invitrogen) supplemented with 10% (vol/vol) fetal bovine serum (FBS) and analyzed on a BD LSRII flow cytometer. For each sample, transfections and flow cytometry measurements were performed in duplicate. Transfection of human cells and isolation of genomic DNA To assess the on-target and off-target indel mutations induced by RGNs targeted to endogenous human genes, plasmids were transfected into U2OS.EGFP or HEK293 cells using the following conditions: U2OS.EGFP cells were transfected using the same conditions as for the EGFP disruption assay described above. HEK293 cells were transfected by seeding them at a density of 1.65 105 א cells per well in 24 well plates in Advanced DMEM (Life Technologies) supplemented with 10% FBS and 2 mM GlutaMax (Life Technologies) at 37°C in a CO2 incubator, After 22 - 24 hours of incubation, cells were transfected with 125 ng of gRNA expression plasmid or an empty U6 promoter plasmid (as a negative control), 375 ng of Cas9 expression plasmid (pJDS246) (Example 1 and Fu et al., 2013), arid 10 ng of a td-Tomato expression plasmid, using Lipofectamine LTX reagent according to the manufacturer ’s instructions (Life Technologies). Medium was changed 16 hours after transfection. For both types of cells, genomic DNA was harvested two days post- transfection using an Agencourt DNAdvance genomic DNA isolation kit (Beckman) according to the manufacturer ’s instructions. For each RGN sample to be assayed, 12 PCT/US2014/029068 WO 2014/144592 individual 4D transfection replicates were performed, genomic DNA was isolated from each, of these 12 transfections, and then these samples were combined to create two "duplicate " pools each consisting of six pooled genomic DNA samples. Indel mutations were then assessed at on-target and off-target sites from these duplicate samples by T7EI assay, Sanger sequencing, and/or deep sequencing as described below.To assess frequencies of precise alterations introduced by HDR with ssODN donor templates, 2xl05 U2OS.EGFP cells were transfected 250 ng of gRNA expression plasmid or an empty U6 promoter plasmid (as a negative control), 750 ng Cas9 expression plasmid (pJDS246), 50 pmol of ssODN donor (or no ssODN for controls), and 10 ng of td-Tomato expression plasmid (as the transfection control). Genomic DNA was purified three days after transfection using Agencourt DNAdvance and assayed for the introduction of a BamHY site at the locus of interest as described below. All of these transfections were performed in duplicate.For experiments involving Cas9 nickases, 2 x 105 U2OS.EGFP cells were transfected with 125 ng of each gRNA expression plasmid (if using paired gRNAs) or 250 ng of gRNA expression plasmid (if using a single gRNA), 750 ng of Cas9-D10A nickase expression plasmid (pJDS271), 10 ng of td-Tomato plasmid, and (if performing HDR) 50 pmol of ssODN donor template (encoding the BamHY site). All transfections were performed in duplicate. Genomic DNA harvested two days after transfection (if assaying for indel mutations) or three days after transfection (if assaying for HDR/ssODN-mediated alterations) using the Agencourt DNAdvance genomic DNA isolation kit (Beckman). T7EI assays for quantifying frequencies of indel mutations T7EI assays were performed as previously described (Example 1 and Fu et al., 2013). In brief, PCR reactions to amplify specific on-target or off-target sites were performed with Phusion high-fidelity DNA polymerase (New England Biolabs) using one of the two following programs: (1) Touchdown PCR program [(98°C, 10 s; 72- 62°C, -1 °C/cycle, 15 s; 72°C, 30 s) x 10 cycles, (98°C, 10 s; 62°C, 15 s; 72°C, 30 s) x25 cycles] or (2) Constant Tm PCR program [(98°C, 10 s; 68°C or 72°C, 15 s; 72°C, 30 s) x 35 cycles], with 3% DMSOor 1 M betaine if necessary. All primers used for these amplifications are listed in Table E.Resulting PCR products ranged in size from 300 to 800 bps and were purified by Ampure XP beads (Agencourt) according to the manufacturer ’s instructions. 200ng of purified PCR products were WO 2014/144592 PCT/US2014/029068 hybridized in 1 x NEB buffer 2 in a total volume of 19 pl and denatured to form heteroduplexes using the following conditions: 95 °C, 5 minutes; 95 to 85 °C, -°C/s; 85 to 25 °C, -0.1 °C/s; bold at 4 °C. 1 ,111 of T7 Endonuclease T (New England Biolabs, 10 units/ul) was added to the hybridized PCR products and incubated at37°C for 15 minutes. The T7EI reaction was stopped by adding 2 pl of 0.25 M EDTAsolution and the reaction products were purified using AMPure XP beads (Agencourt) with elution in 20 pl O.lxEB buffer (QIAgen). Reactions products were then analyzed on a QIAXCEL capillary electrophoresis system and the frequencies of indel mutations were calculated using the same formula as previously described (Reyon etal., 2012).

TABLE E Publicatio n ID Expected Off-Target Sequences (Expected) - HS GRCh37 SEQ ID NO: Mismatche s in target compared to on- target site Actual Target in U2OS.EGFP cellsForward PCR PrimerSEQ ID NO:Reverse PCR PrimerSEQ ID NO:PCR Condi tions Wats on- Cric kTran sver sion s non- Watso n- Crick Trans -ver- sions Tran- sitio ns Target 1GGGTGGGGGGAGTTTGCTCCTGG1269. 0TCCAGATGGCACA TTGTCAG1270 .AGGGAGCA GGAAAGTG AGGT1271. DMSO OT1-1GGGTGGGGGGAG TTTGCCCCAGG1272. 1GGGGCCCACTCTTCTTCCAT1273 ,ACCCAGACTCCTGGTGTGGC1274 .NoDMSO0 1 oo OT1-2GCGTGGGGGGTGTTTGCTCCCGG1275. 2GCTAAGCAGAGATGCCTATGCC1276.A.CCA.CCCTTTCCCCCAGAAA1277 . DMSO 2 0 0 o OT1-3GGATGGAGGGAGTTTGCTCCTGG1278. 2ACCCCACAGCCAG GTTTTCA1279 .GAATCACTGCACCTGGCCATC1280 . DMSO 0 0 2 0T1-4GGGAGGGTGGAG TTTGCTCCTGG1281. 2TGC GGCAAC T T CAGACAACC1282 ,TAAAGGGCGTGCTGGGAGAG1283 . DMSO 1 1 0 OT1-5GGGTGGGTGGAG TTTGCTACTGG1284. 2GCATGTCA.GGATCTGACCCC1285 ,TGCAGGGCCATCTTGTGTGT1286 . DMSO 0 2 0 OT1-6CGGGGGAGGGAG TTTGCTCCTGG1287. 3CCACCACATGTTCTGGGTGC1288 .CTGGGTCT GTTCCCTG TGGG1289. DMSO 1 1 1 OT1-7GAGTGGGTGGAGTTTGCTACAGG1290. 3GGCTCTCCCTGCCCTAGTTT1291 ,GCAGGTCAAGTTGGAACCCG1292 . DMSO 0 2 1 OT1-8GGGAGGGGAGAG TTTGTTCCAGG1293. 3GGGGCTGAGAACA CA.TGAGATGCA1294 .AGATTTGT GCACTGCC TGCCT1295. DMSO 1 0 2 W O 2014/144592 PCT/US2014/029068 TABLE E OT1-9GGGAGGGGGCAGGTTGCTCCAGG1296. 3CCCGACCTCCGCTCCAAAGC12S7 ,GGACCTCT GCACACCC TGGC1290 . DMSO 2 1 0 OT1-10GGGAGGGGGGAGTGT3TTCCGGG1299.־כ.TGCAAGGTCGCATAGTCCCA1300 ,CAGGAGGG GGAAGTGT GTCC1301. DMSO 1 1 1 OT1-11G GGGAG GG GAAG TTTGCTCCTGG1302.GCCCATTCTTTTTGCAGTGGA1303 ,GAGAGCAA GTTTGTTC CCCAGG1304 . DMSO 0 1 2 OT1-12GGGGGTGGGGAC TITSCTCCAGG1305.GCCCCCAGCCCCT CTGTTTC1306.GCTGCTGG TAGGGGAG CTGG1307 . DMSO 1 2 D OT1-13GGGTCGGGGGAG TGGGCTCCAGG1308. 3CGGCTGCCTTCCCTGAGTCC1309 ,GGGTGACGCTTGCCATGAGC1310 .72C Annea 1, 3% DMSO2 0 OT1-14GGGTGGCTGGAGTTTGCTGCTGG1311. 3TGACCCTGGAGTACAAAATGTTCCCA1312 ,GCTGAGAC AJkCCAGCC CAGCT1313 .72C Annea 2, 3% DMSO1 0 OT1-15GGGTGGGGGGTGCCTGCTCCAGG1314.TGCCTCCACCCTTAGCCCCT1315 ,GCAGCCGA TCCACACT GGGG1316. DMSO 1 0 2 OT1-16GGTTGAGGGGAGTCT3CTCCAGG1317. 3AACTCAGGACAACACTGCCTGT1318 .CCCAGGAGCAGGGTACAATGC1319 . DMSO 0 1 2 OT1-17GTGTGGGTGGCG TTTGCTCCAGG1320. 3TCCTCCTTGGAGAGGGGCCC1321,CCTTGGAAGGGGCCTI GGTGG1322 . DMSO 0 3 0 OT1-18AGGTGGTGGGAG CTTGTTCCTGG1323. 4CCGAGGGCATGGGCAATCCT1324 ,GGCTGCTG CGAGTTGC CAAC1325. DMSO 0 1 3 OT1-19AGTTTGGGGGAG TTTGCCCCAGG1326 . 4TGCTTTGCATGGGGTCTCAGACA1327 .GGGTTGCTTGCCCTCTGTGT1328 . DMSO 0 2 2 ATGTGTGGGGAATTTGCTCCAGGAGCTCCTTCTCAT CACAGAAGOT1-201329 .TTCTCTTCTGCTG T1330 . GATGTGTGCAGGTT1331. DMSO 0 2 2 OT1-21CAGTGGGGGGAGCTTTCTCCTGG1332. 4AGCAGACACAGGTGAATGCTGCT1333.GGTCAGGTGTGCTGCTAGGCA1334 . DMSO 1 1 2 W O 2014/144592 PCT/US2014/029068 TABLE E OT1-22GAGGGGGAGCAG TTTGCTCCAGG1335. 4CCTGTGGGGCTCT CAGGTGC1336 ,ACTGCCTGCCAAAGTGGGTGT1337 .No DMSOTD1 2 OT1-23GGAGGAGGGGAG TCTGCTCCAGG1338. 4AGCTGCACTGGGGAATGAGT1339.TGCCGGGT AATAGCTG GCTT1340. DMSO 0 1 372C OT1-24GGAGGGGGGGCT TTTGCTCCAGG1341. 4CCAGCCTGGGCAACAAAGCG1342 ,GGGGGCTT CCAGGTCA CAGG1343.Annea 1, 3% DMSO, 6%3 1 DMSOOT1-25GGGCAAGGGGAGGTTGCTCCTGG1344. 4TACCCCCACTGCCCCATTGC1345 .ACAGGTCCATGCTTAG CAGAGGG1346. DMSO 0 1 3GGGTGATTGAAGTT OT1-26GGGTGATTGAAGTTTGCTCCAGG1347. 4TGCICCAGG (SEQID NO:2225)GGGTGATTGAAGTTACGGATTCACGAC GGAGGTGC1348 .CCGAGTCCGTGGCAGA 1349 . DMSO/2 2TGCIGCAGG (SEQID NO:2226)OT1-27GGGTGTGGGGTC ATTGCTCCAGG1350 . 4TGTGGTTGAAGTAGGGGACAGGT1351.TGGCCCAA TTGGAAGT GATTTCGT1352. DMSO 3 1 0 OT1-28GGTGGGGGTGGG TTTGCTCCTGG1353 . 4TGGGATGGCAGAGTCATCAACGT1354 .GGCCCAAT CGGTAGAG GATGCA1355 . DMSO 0 3 1 OT1-29GTGGGGGTAGAGTTTGCTCCAGG1356. 4ATGGGGCGCTCCAGTCTGTG1357 .TGCACCCA CACAGCCA GCAA1358 . DMSO 0 3 1 OT1-30TAGTGGAGGGAGGTTGCTCCTGG1359. 4GGGGAGGGAGGACCAGGGAA1360 .AATTAGCTGGGCGCGG TGGT1361.72C Annea 1, 3% DMSO1 3 OT1-31TGCTCGGGGGAGTTTGCACCAGG1362. 4ATCCCGTGCAGGAAGTCGCC1363.CAGGCGGC CCCTTGAG GAAT1364 . DMSO 3 1 0 OT1-32TGGAGAGGGGAGTTGGCTCCTGG1365. 4CCCCAACCCTTTG CTCAGCG1366.TGAGGAGA ACACCACA GGCAGA1367 . DMSO 1 2 1 W O 2014/144592 PCT/US2014/029068 TABLE E OT1-33TGGTGTTGGGAGTCTGCTCCAGG1368 . 4ATCGACGAGGAGG GGGCCTT1369 .CCCCTCAC TCAAGCAG GCCC1370 . DMSO 0 3 1 OT1-34TTGGGGGGGCAGTTTGCTCCTGG1371. 4TGCTCAAGGGGCC TGTTCCA1372 .CAGGGGCA GTGGCAGG AGTC1373 .NoDMSO3 0 OT1-35AAGTAAGGGAAGTTTGCTCCTGG1374.TGCCTGGCACGCA GTAGGTG1375 .GGGAAGGG GGAACAGG TGCA1376. DMSO 0 0 5 OT1-36AGAAGAGGGGATTTTGCTCCTGG1377. 5 Not optimized 1 1 3 OT1-37ATCTGGGGTGATTTTGCTCCTGG1378. 5ACCTGGGCTTGCCACTAGGG1379 .GCTGCTCG CAGTTAAG CACCA1380 . DMSO 1 3 1 OT1-38CTCTGCTGGGAGTTTGCTCCTGG1381. 5GTGGCCGGGCTAC TGCTACC1382 ,GGTTCCACAAGCTGGGGGCA1383. DMSO 3 2 0 OT1-39CTGGTGGGGGAG CTTGCTCCAGG1384. 5 Not optimized 1 3 1wOT1-40CTTTCGGGGGAG TTTGCGCCGGG1385 . 5GCAAGAGGCGGAGGAGACCC1386 ,AGAGTCAT CCATTTCC TGGGGGC1387 . DMSO 2 3 0AGGGAATC IM0T1-41CTTTGGGGTTAGTTTGCTCCTGG1388. 5GGGGTCAGTGGTGATATCCCCCT1389 ,CTTTTTCCATTGCTTG1390 .beta!ne,4 0TTT TDOT1-42GCTCTGGGGTAGTCTGCTCCAGG1391.AGAGAGGCCACGT GGAGGGT1392 .GCCTCCCC TCCTCCTT CCCA1393 . DMSO 1 3 1 OT1-43GTCTCTCGGGAG- TTTGCTCCGGG1394. 5GACAGTGCCTTGC GATGCAC1395 .TCTGACCG GTATGCCT GACG1396. DMSO 3 2 0 OT1-44TCCTGAGGGCAGTTTGCTCCAGG1397. 5TGTGTGAACGCAGCCTGGCT1398.TGGTCTAG TACTTCCT CCAGCCTT1399. DMSO 3 1 1 OT1-45TCTTTGGGAGAGTTTGCTCCAGG1400 . 5GGTTCTCCCTTGG CTCCTGTGA1401.CCCACTGC TCCTAGCC CTGC1402 . DMSO 1 3 1 W O 2014/144592 PCT/US2014/029068 TABLE E O OT1-46ACAACTGGGGAGTTTGCTCCTGG1403. 6TGAAGTCAACAATCTAAGCTTCCACCT1404 ,AGCTTTGGTAGTTGGA GTCTTTGA AGG1405 . DMSO 3 1 2 OT1-47ACAAGGTGGAAGTTTGCTCCTGG1406. 6TGATTGGGCTGCAGTTCATGTACA1407 .GCACAGCC TGCCCTTG GAAG1408 . DMSO 2 1 3 011-48ACATAGAAGGAGTTTGCTCCAGG1409. 6TCCATGGGCCCCTCTGAAAGA1410 .AGCGGCTT CTGCTTCT GCGA1411. DMSO 1 0 5 OT1-49AGACCCAGGGAGTTTGCTCCCGG1412. 6GCGGTTGGTGGGGTTGATGC1413 ,GAGTTCCT CCTCCCGC CAGT1414 . DMSO 2 0 4 OT1-50AGACCCAGGGAGTTTGCTCCCGG1415. 6AGGCAAGATTTTCCAGTGTGCAAGA1416,GCTTTTGCCTGGGACTCCGC1417 . DMSO 2 0 4 OT1-51CACGGAGGGGTGTTTGCTCCTGG1418. 6GCTGCTGGTCGGGCTCTCTG1419 .GCTCTGTCCCACTTCC CCTGG1420 .No DMSOTD1 2op0T1-52CAGAGCTTGGAGTTTGCTCCAGG1421. 6GCTGCGAGGCTTCCGTGAGA1422 .CGCCCCTA GAGCTAAG GGGGT1423 . DMSO 3 2 1 OT1-53CrATTGATGGAGTTTGCTCCTGG1424. 6CCAGGAGCCTGAGAGCTGCC1425 .AGGGCTAG GAC T G C AG TGAGC1426 . DMSO 1 3 2 OT1-54CTTTCTAGGGAGTTTGCTCCTGG1427. 6CTGTGCTCAGCCTGGGTGCT1428 ,GCCTGGGGCTGTGAGT AGTIT1429 . DMSO 2 3 1 OT1-55GCCATGCTGGAGTTTGCTCCAGG1430. 6AGCTCGCGCCAGATCTGTGG1431,ACTTGGCA GGCTGAGG CAGG1432 .72C Annea 1, 3% DMSO2 0 1433. 1434 , 1435 .Target 2GACCCCCTCCACCCCGCCTCCGG1436 . 0AGAGAAGTCGAGG AAGAGAGAG1437,CAGCAGAAAGTTCATGGTTTCG1438 . DMSO OT2-1GACCCCCCCCAC CCCGCCCCCGG1439. 2TGGACAGCTGCAG TACTCCCTG1440 ,ACTGATCGATGATGGC CTATGGGT1441. DMSO 0 0 2 2014/144592 PCT/US2014/029068 TABLE E 2 ־ 012GGGCCCCTCCACCCCGCCTCTGG1442 . 2CAAGATGTGCACTTGGGCTA1443 ,GCAGCCTA TTGTCTCC TGGT1444 . DMSO 1 0 1 OT2-3AACCCCATCCACCCGGCCTCAGG1445 . 3GTCCAGTGCCTGACCCTGGC1446 ,AGCATCAT GCCTCCAG CTTCA1447. DMSO 1 1 1 012-4CACCCCCTCAACACCGCCTCAGG1448. 3GCTCCCGATCCTCTGCCACC1449 ,GCAGCTCC CACCACCC TCAG1450 . DMSO 1 2 0 OT2-5CACCCCCTCCCC TCCGCCTCAGG1451. 3GGGGACAGGCAGGCAAGGAG1452 .GTGCGTGTCCGTTCACCCCT1453 . DMSO 1 1 1 012-6CTACCCCTCCACCCCGCCTCCGG1454 .AAGGGGCTGCTGGGTAGGAC1455 .CGTGATTCGAGTTCCTGGCA1456 . DMSO 2 1 0 012-7GACCCGCCCCGCCCCGCCTCTGG1457 . 3GACCCTCAGGAAG CTGGGAG1458 ,CTGCGAGATGCCCCAAATCG1459 .IM beta! ne,TD0 2*Zb012-8GATCGACTCCA.CCCCGCCTCTGG1460 . 3CCGCGGCGCTCTGCTAGA1461 .TGCTGGGA TTACAGGC GCGA1462 . DMSO 1 1 1 012-9GCCCCCACCCAC CCCGCCTCTGG1463. 3CCAGGTGGTGTCAGCGGAGG1464 ,TGCCTGGCCCTCTCTG AGTCT1465 . DMSO 0 2 1 0T2-1OGCCCCGCTCCTC CCCGCCTCCGG1466. 3CGACTCCACGGCGTCTCAGG1467 ,CAGCGCAG TCCAGCCC GATG1468 .IM beta! ne, TD1 0 0T2-11GGGCCCCTCCAC CAGGCCTCAGG1469. 3CTTCCCTCCCCCA GCACCAC1470 .GCTACAGGTTGCACAGTGAGAGGT1471. DMSO 1 1 1 012-12GGCCCCCTCCTCCTCGCCTCTGG1472. . 3CCCCGGGGAGTCTGTCCTGA1473 ,CCCAGCCGTTCCAGGT CTTCC1474 .72C Annea 1, 3% DMSO0 2 0T2-13GGCGCCCTCCACCCTGCCTCGGG1475. 3GAAGCGCGAAAACCCGGCTC1476 ,TCCAGGGT CCTTCTCG GCCC1477 . DMSO 1 0 2 W O 2014/144592 PCT/US2014/029068 TABLE E OT2-14GTCCTCCACCAC CCCGCCTCTGG1478. 3AGGGTGGTCAGGGAGGCCTT1479 ,CATGGGGC TCGGACCT CGTC1480 . DM50 2 0 1 OT2-15TACCCCCCACACCCCGCCTCTGG1481.GGGAAGAGGCAGGGCTGTCG1482TGCCAGGA AGGAAGCT GGCC1483 .72C Aime a 1, 3% DMSO2 1 OT2-16AACCCATTCCACCCrGCCTCAGG1484. 4GAGTGACGATGAG CCCCGGG1485 .CCCTTAGC TGCAGTCG CCCC1486 .68C Amnea 1, 3% DMSO1 3 OT2-17ACACCCCCCCACCCCGCCTCAGG1487. 4CCCATGAGGGGTT TGAGTGC1488 ,TGAAGATG GGCAGTTTGGGG1489 . DMSO 0 2 2 OT2-18AGCCCCCACCTCCCC3CCTCGGG1490. 4CACCTGGGGCATCTGGGTGG1491,ACTGGGGTTGGGGAGG GGAT1492 . DMSO 2 0 2 OT2-19ATTCCCCCCCACCCCGCCTCAGG1493. 4TCATGATCCCCAAAAGGGCT1494 .CCATTTGTGCTGATCTGTGGGT1495 . DMSO 1 0 3 OT2-20CCCCACCCCCACCCCGCCTCAGG1496. 4TGGTGCCCAGAATAGTGGCCA1497 .AGGAAATG TGTTGTGC CAGGGC1498 . DMSO 1 2 1 OT2-21CCCCCCCACCACCCCGCCCCGGG1499. 4GCCTCAGACAACCCTGCCCC1500 .GCCAAGTG TTACTCAT CAAGAAAG TGG1501.NoDMSOTD1 1 OT2-22CCCCCCCCCCCC CCCGCCTCAGG1502. 4GCCGGGACAAGACTGAGTTGGG1503 ,TCCCGAACTCCCGCAAAACG1504 . DMSO 1 2 1 OT2-23CGCCCTCCCCACCCCGCCTCCGG1505. 4TGCTGCAGGTGGTTCCGGAG1506,CTGGAACC GCATCCTC CGCA1507 .NoDMSOCD0 3 OT2-24CTCCCCACCCACCCCGCCTCAGG1508 . 4ACACTGGTCCAGGTCCCGTCT1509 .GGCTGTGC CTTCCGAT GGAA1510 . DMSO 2 1 1 OT2-25CTCTCCCCCCACCCCGCCTCTGG1511. 4CTCTCCCCCCACCCCCCCTCTGG (SEQID MO:2227)ATCGCGCCCAAAGCACAGGT1512 ,AGGCTTCTGGAAAAGT CCTCAATG CA1513 . DMSO -2> 0 2 W O 2014/144592 PCT/US2014/029068 TABLE E OT2-26GCCTCTCTGCACCCCGCCTCAGG1514. 4 Not optimized 1 1 2 OT2-27GTCACTCCCCAC CCCGCCTCTGG1515. 4CCCTCATGGTGGTCTTACGGCA1516.AGCCACACA.TCTTTCTGGTAGGG1517 . DMSO 1 1 2 OT2-28TGCCCCCTCCCCCCAGCCTCTGG1518. 4TGCGTCGCTCATGCTGGGAG1519 ,AGGGTGGGGTGTACTG GCTCA1520 . DMSO 0 3 1 OT2-29TGCCCCTCCCACCCCGCCTCTGG1521. 4GAGCTGAGACGGCACCACTG1522 .TGGCCTTGAACTCTTGGGCT1523 .IM betai ne, CD1 3 012-30TTCCCCTTCCACCCAGCCTCTGG1524. 4 Not optimized 1 2 1 OT2-31TTCTCCCTCCTCCCCGCCTCGGG1525. 4AGTGAGAGTGGCA CGAACCA1526CAGTAGGT GGTCCCTT CCGC1527 . DMSO 2 1 1 OT2-32ACCCTCGCCCACCCCGCCTCAGG1528. 5 Not optimized 1 1 3 OT2-33AGCCAACCCCACCCCGCCTCTGG1529. 5GGGAGAACCTTGTCCAGCCT1530,AAGCCGAA AAGCTGGG CAAA1531. DMSO 0 2 3 OT2-34AGGCCCCCACACCCCGCCTCAGG1532.CTTCCCAGTGTGGCCCGTCC1533,ACACAGTCA.GAGCTCCGCCG1534. DMSO 1 1 3 OT2-35AGGCCCCCCCGCCCCGCCTCAGG1535. 5 Net optimized 1 0 4 OT2-36ATC’TGCCACCACCCCGCCTCGGG1536. 5C T GAGAGG G GGAG GGGGAGG1537 ,TCGACTGGTCTTGTCC TCCCA1538 .58C Anne a 1, 3% DMSO0 2 OT2-37CATCTTCCCCACCCCGCCTCTGG1539. 5C AGC C TGCT GC AT CGGAAAA1540TGCAGCCAAGAGAAAAAGCCT1541.IM betai ne, TD0 4 OT2-38CTTTCCCTCCACCCAGCCTCTGG1542. 5TCCCTCTGACCCG GAACCCA1543,ACCCGACTTCCTCCCC ATTGC1544 . DMSO 2 1 OT2-39GTCGAGGTCCACCCCGCCTCAGG1545. 5TGGGGGTTGCGTGCTTGTCA1546,GCCAGGAG GACACCAG GACC1547 . DMSO 4 1 0 W O 2014/144592 PCT/US2014/029068 TABLE E OT2-40GTCGAGGICCACCCCGCCTCAGG1548. 5ATCAGGTGCCAGGAGGACAC1549 ,GGCCTGAG AGTGGAGA GTGG1550. DMSO 4 1 0 OT2-41TCAGACCTCCAO CCCGCCTCAGG1551. 5 Not optimized 1 4 0A.CCTCTCCOT2-42TGCAACCICCTC CCCGCCTCGGG1552. 5TGAGCCACATGAA TCAAGGCCTCC1553 ,AAGTCTCAGTAACTCT1554 . DMSO 1 3 1CTOT2-43ACCAGTCTGCACCCCGCCTCTGG1555. 6GGTCCCTCTGTGCAGTGGAA1556 .CTTTGGTG GACCTGCA CAGC1557 . DMSO 2 2 2 OT2-44ACTACCCACCTCCCCGCCTCAGG1558. 6GCGAGGCTGCTGACTTCCCT1559 ,GCTGGGAC TACAGACA TGTGCCA1560 . DMSO 2 2 2 OT2-45ATTTCCCCCCCCCCCGCCTCAGG1561. 6ATTICCTCCCCCCC c-CCTCAGG (SEQ ID NO:2228)ATTGCAGGCGTGT CCAGGCA1562 .AAATCCTG CATGGTGA TGGGAGT1563 . DMSO 1 1 5 OT2-46CCACCATCCCACCCCGCCTCTGG1564 . 6TGCTCTGCCATTT ATGTCCTATGAAC T1565 ,ACAGCCTC TTCTCCAT GACTGAGC1566. DMSO 1 3 2soסס OT2-47CCCAAGOCCCACCCCGCCTCGGG1567. 6TCCGCCCAAACAG GAGGCAG1568 .GCGGTGGGGAAGCCATTGAG1569 . DMSO 2 3 1 OT2-48CCGCGCTTCCGCCCCGCCTCTGG1570. 6GGGGGTCTGGCTC ACCTGGA1571.CCTGTCGG GAGAGTGC CTGC1572 . DMSO 3 1 2 OT2-49CCTGCCATGCACCCCGCCTCAGG1573. 6TCCTGGTTCATTTGCTAGAACTCTGGA157 4 .ACTCCAGATGCAACCAGGGCT1575 . DMSO 3 2 1 OT2-50CTGCC’TCCTCACCCCGCCTCAGG1576. 6CGTGTGGTGAGCC TGAGTCT1577 .GCTTCACC GTAGAGGC TGCT1578 . DMSO0 3TCAGTGACOT2-51TCITCTTICCACCCCGCCTCAGG1579. 6AGGCCCTGATAAT TCATGCTACCAA1580.AACCTTTTGTATTCGG1581. DMSO 0 2dCA012-52TTGACCCCCCGCCCCGCCTCAGG1582. 6 Not optimized 2 2 2 W O 2014/144592 PCT/US2014/029068 TABLE E Target 3GGTGAGTGAGTG TGTGCGTGTGG1503. 0TCCAGATGGCACATTGTCAG1584 ,AGGGAGCA GGAAAGTG AGGT1585 . DMSO OT3-1GGTGAGTGAGTGTGTGTGTGAGG1586. 1GCAGGCAAGCTGTCAAGGGT1587 .CACCGACACACCCACTCACC1588 . DMSO 0 0 1 OT3-2AGTGAGTGAGTGTGTGTGTGGGG1589. 2GAGGGGGAAGTCA CCGACAA1590 ,TACCCGGGCCGTCTGTTAGA1591. DMSO 0 0 2 OT3-3AGTGTGTGAGTG TGTGCGTGTGG1592. 2GACACCCCACACACTCTCATGC1593 .TGAATCCCTTCACCCCCAAG1594 . DMSO 1 0 1 OT3-4GGTGAGTGAGTGTATGCGTGTGG1595. 2TCCTTTGAGGTTCATCCCCC1596.CCAATCCAGGATGATTCCGC1597 . DMSO 1 0 1 OT3-5GGTGAGTGAGTGTGTGAGTGAGG1598. 2CAGGGCCAGGAACACAGGAA1599 .GGGAGGTA TGTGCGGG AGTG1600 . DMSO 1 1 0 OT3-6GGTGAGTGAGAG TGTGTGTGTGG1601. 2TGCAGCCTGAGTG AGCAAGTGT1602 ,GCCCAGGT GCTAAGCC CCTC1603 . DMSO 1 0 1 se OT3-7GGTGAGTGAGTG AGTGAGTGAGG1604. 2TACAGCCTGGGTGATGGAGC1605 .TGTGTCATGGACTTTC CCATTGT1606.IM beta! ne, TD1 0 OT3-8GGTGAGTGAGTGAGTGAGTGAGG1607. 2GGCAGGCATTAAACTCATCAGGTCC1608 .TCTCCCCCAAGGTATCAGAGAGCI1609 . DMSO 1 1 0 OT3-9GGTGAGTGAGTG CGTGCGGGTGG1610. 2GGGCCTCCCTGCT GGTTCTC1611.GCTGCCGTCCGAACCCAAGA1612 . DMSO 0 1 1 OT3-10GGTGAGTGTGTGTGTGAGTGTGG1613. 2ACAAACGCAGGTGGACCGAA1614 ,ACTCCGAA AATGCCCC GCAGT1615 . DMSO 1 1 0 OT3-11GGTGAGTGTGTGTGTGCATGTGG1616. 2AGGGGAGGGGACATTGCCT1617 .TTGAGAGGGTTCAGTGGTTGC1618 . DMSO 1 0 1 OT3-12GGTGTGTGAGTG TGTGTGTGTGG1619. 2CTAAIGCTTACGG CTGCGGG1620AGCCAACG GCAGATGC AAAT1621. DMSO 1 0 1 W O 2014/144592 PCT/US2014/029068 TABLE E 0T3-13GGTGTGTGTGTG TGTGCGTGCGG1622. 2GAGCGAAGTTAACCCACCGC1623 .CACACATGCACATGCCCCTG1624 .680, 3%DMSO0 0 OT3-14GGTGTGTGTGTG TGTGCGTGTGG1625. 2GCATGTGTCTAAC TGGAGACAATAGCA1626TCCCCCATATCAACACACACA1627 . DMSO 2 0 0 OT3-15GGTGTGTGTGTG TGTGCGTGTGG1628. 2GCCCCTCCCGCCTTTTGTGT1629.TGGGCAAAGGACATGAAACAGACA1630 . DMSO 2 0 0ACGAACAGOT3-16GGTGTGTGTGTG TGTGCGTGTGG1631.GCCTCAGCTCTGC TCTTAAGCCC1632 .ATCATTTTTCATGGCT1633 . DMSO 2 0 0TCCOT3-17GTTGAGTGAATGTGTGCGTGAGG1634. 2CTCCAGAGCCTGG CCTACCA1635:CCCTCTCC GGAAGTGC CTTG1636 . DMSO 0 1 1 OT3-18TGTGGGTGAGTGTGTGCGTGAGG1637. 2TCTGTCACCACAC AGTTACCACC1638 .GTTGCCTG GGGATGGG GTAT1639 . DMSO 0 1 1 OT3-19ACTGTGTGAGTGTGTGCGTGAGG1640. 3GGGGACCCTCAAGAGGCACT1641.GGGCATCA AAGGATGG GGAT1642 . DMSO 2 0 1 OT3-20AGAGAGTGAGTGTGTGCATGAGG1643. 3TGTGGAGGGTGGGACCTGGT1644 .ACAGTGAGGTGCGGTCTTTGGG1645 . DMSO 1 0 2 OT3-21AGCGAGTGGGTGTGTGCGTGCGG1646. 3CGGGGTGGCAGTGACGTCAA1647 ,GGTGCAGT CCAAGAGC CCCC1648 . DMSO 0 0 3 OT3-22AGGGAGTGACTG TGTGCGTGTGG1649. 3AGCTGAGGCAGAGTCCCCGA1650 ,GGGAGACAGAGCAGCGCCTC1651. DMSO 1 1 1 OT3-23AGTGAGTGAGTGAGTGAGTGAGG1652. 3ACCACCAGACCCOACCTCCA1653 .AGGACGAC TTGTGCCC CATTCA1654 .72C Annea 1, 3% DMSO1 1 OT3-24CATGAGTGAGTG TGTGGGTGGGG1655. 3GGGTCAGGACGCAGGTCAGA1656,TCCACCCACCCACCCATCCT1657.72C Annea 1, 3% DMSO0 1 W O 2014/144592 PCT/US2014/029068 TABLE E OT3-25CGTGAGTGTGTGTAT3CGTGTGG1658 . 3ACACTCTGGGCTAGGTGCTGGA1659 ,GCCCCCTCACCACATGATGCT1660 . DMSO 2 0 1 013-26GGACTGTGAGTGTGTGCGTGAGG1661. 3GGGGCCATTCCTCTGCTGCA1662 .TGGGGATCCTTGCTCATGGC1663 . DMSO 3 0 0 OT3-27GGTGTGTGCCTG TGTGCGTGTGG1664. 3ACACACTGGCTCGCATTCACCA1665 .CCTGCACGAGGCCAGG TGTT1666. DMSO 2 1 0 OT3-28GTTTCATGAGTG TGTGCGTGGGG1667. 3TGGGCACGTAGTAAACTGCACCA1668.CTCGCCGCCGTGACTG TAGG1669 . DMSO 0 3 1 OT3-29TGAGTGTGAGTGTGTGCGTGGGG1670. 3TCAGCTGGTCCTGGGCTTGG1671.AGAGCACTGGGTAGCAGTCAGT1672 . DMSO 2 1 0 OT3-30TGCCAGTGAGTGTGTGCGTGTGG1673. 3AGACACAGCCAGG GCCTCAG1674 .GGTGGGCG TGTGTGTG TACC1675 .68C, 3%DMSO1 1 OT3-31TGGGTGTGAGTGTGTGCGTGTGG1676. 3ACACTCTCACACACGCACCAA1677 .GAGAAGTC AGGGCTGG CGGG1678.72C Anne a 1, 3% DMSO2 0 OT3-32TGTATGTGAGTGTGTGCGTGTGG1679. 3ACTGCCTGCATTTCCCCGGT1680 .TGGTGAGGGCTTCAGGGAGC1681. DMSO 1 1 1 OT3-33TGTGAGAGAGAGTGTGCGTGTGG1682. 3GCCAGGTTCATTGACTGCCC1683 .TCCTTCTACACATCGGCGGC1684 . DMSO 2 1 0 OT3-34TGTGCCTGAGTGTGTGCGTGTGG1685. 3CGAGGGAGCCGAGTTCGTAA1686 .CTGACCTG GGGCTCTG GTAC1687 . DMSO 1 2 D OT3-35TGTGTGTGTGTG TGTGCGTGTGG1688. 3TCCTCGGGAAGTC ATGGCTTCA1689 .GCACTGAGCAACCAGGAGCAC1690 . DMSO 2 1 0 OT3-36AGCGTGTGAGTGTATGCGTGGGG1691. 4 Not optimized 1 0 3 OT3-37ATTGAGTGTGTG AGTGCGTGGGG1632. 4TAAACCGTTGCCCCCGCCTC1693 .GCTCCCCT GCCAGGTG AACC1694 . DMSO 2 1 1 W O 2014/144592 PCT/US2014/029068 TABLE E OT3-36CATGTGTGGGTG TGTSCGTGTGG1695. 4CCTGCTGAGACTC CAGGTCC1696.CTGCGGAG TGGCTGGC TATA1697 . DMSO 2 0 2 OT3-39CCCGAGTGTGTGTGTSCGTGTGG1698. 4CTCGGGGACTGAC AAGCCGG1699.GGAGCAGC TCTTCCAG GGCC1700 . DMSO 3 0 1 OT3-40' CTGGAGTGAGTGTGTGTGTGTGG1701. 4CCCCGACCAAAGC AGGAGCA1702 .CTGGCAGCCTCTGGATGGGG1703 . DMSO 1 2 1 OT3-41GTTTCAIGAGTGTGTGCGTGGGG1704. 4 Not optimized 0 3 1 OT3-42TATGTGTGCGTGTGTGCGTGTGG1705. 4ATTTCAGAGCCCCGGGGAAA1706.AGGCCGCG GTGTTATG GTTA1707 . DMSO 1 2 1TGACATATOT3-43TATGTGTGTGTG TGTSCGTGGGG1708. 4GCCAGTGGCTTAGTGTCTTTGTGT1709 .TTTCCTGGGCCATGGG1710 . DMSO 2 1 1TCCATGCTGOT3-44TCTGTGTGTGTGTGTGCGTGGGG1711. 4TGCCAGAAGAACA TGGGCCAGA1712 .ACATCATATACTGGGA1713. DMSO 3 1 0AGCOT3-45TCTGTGTGTGTG TGTGCGTGTGG1714. 4GCGTGTCTCTGTGTGCGTGC1715 ,CCAGGCTG GGCACACA GGTT1716. DMSO 3 1 0 OT3-46TGAGCGTGAGTGTGAGCGTGTGG1717. 4 Not optimized 2 2 0AGGATGAGOT3-47TGTCTTTGAGTGTGTGCGTGTGG1718. 4TGCCCAGTCCAAT ATTTCAGCAGCT1719 .TTCATGTCCTTTGTGG1720 . DMSO 2 2 0GG 013-48TTIGTGTGTGTGTGTGCGTGTGG1721. 4GGGT GAAAATT TGGTACTGTTAGCTG 1722 .AATGACTCA.TTCCCTGGGTATCTC1723 . DMSO 2 2 0CCAOT3-49AAGGCGTGTGTGTGTSCGTGTGG1724. 5TGCCCCATCAATC ACCTCGGC1725 .CAAGGTCG GCAGGGCA GIGA1726. DMSO 1 2 2 OT3-50AATTCGTGTGTGTGTGCGTGGGG1727.GCCTCCTCTGCCGCTGGTAA1728TGAGAGTTCCTGTTGCTCCACACT1729 . DMSO 1 2 2 ؛،< W O 2014/144592 PCT/US2014/029068 TABLE E OT3-51ATGGTGTGTGTGTGTGCGTGTGG1730 . 5 Not optimized 2 2 1 OT3-52CACGTGTGTGTGTGTGCGTGTGG1731. 5GCCACCAAAATAGCCAGCGT1732 .ACATGCAI CTGTGTGT GCGT1733 . DMSO 3 0 2 OT3-53GAAATTTGAGTG TGTGCGTGTGG1734. 5ACAGACTGACCCTTGAAAAATACCAG 1735 .TGTATCTT TCTTGCCA ATGGTTTT1736. DMSO 2 1 2cccOT3-54TAAGTGTGTGTGTGTGCGTGTGG1737 . 5AGCCAAATTTCTCAACAGCAGCACT1738TCCTGGAGAGCAGGCA TTTTTGT1739 . DMSO 3 1 1 OT3-55TATATGTGTGTGTGTGCGTGGGG1740 . 5ACCTCCTTGTGCTGCCTGGC1741 .GGCGGGAA GGTAACCC TGGG1742 . DMSO 2 1 2 OT3-56TATCTGTGTGTGTGTGCGTGTGG1743. 5CACAAAGCTCTAC CTTTCCAGTAGTG T1744TGATCCGATGGTTGTT CACAGCT1745 . DMSO 3 1 1 OT3-57TTTATGTGTGTGTGTGCGTGTGG1746. 5TGTGGGGATTACCTGCCTGGC1747ACGCACAAAAATGCCCTTGTCA1748 . DMSO 2 2 1 OT3-58TTTTTGTGTGTG TGTGCGTGGGG1749 . 5TGAGGCAGACCAGTCATCCAGC1750GCCCGAGCACAGTGTAGGGC1751. DMSO 2 3 0 OT3-59AAAAATTGTGTGTGTGCGTGGGG1752. 6ATTAGCTGGGCGTGGCGGAG1753ACTGCATC TCATCTCA GGCAGCT1754 . DMSO 2 1 3TCAGCTTCOT3-60ACAATGTGTGTGTGTGCGTGTGG1755. 6TGAAGCAGAAGGA GTGGAGAAGGA1756ACATCTGTTTCAGTTC1757 . DMSO 4 0 2AGTOT3-61ATGTGGTGTGTGTGTGCGTGTGG1758. 6TGGTGGAGTGTGTGTGTGGT1759.AGAGCAGAAAGAGAGTGCCCA1760 . DMSO 12 OT3-62CAAAATTGTGTGTGTGCGTGTGG1761. 6GCCCCTGTACGTCCTGACAGC1762 .TGCACAAG CCACTTAG CCTCTCT1763 . DMSO 3 1 2 OT3-63CCCTGGTGTGTGTGTGCGTGTGG1764. 6AGCGCAGGTAAACAGGCCCA1765 .TCTCTCGCCCCGTTTCCTTGT1766 . DMSO 3 1 2 W O 2014/144592 PCT/US2014/029068 TABLE E OT3-64TCCGCTTGTGTG TGTGCGTGGGG1767 . 6ATGGGTGCCAGGTACCACGC1768.ACAGCAGG AAGGAGCC GCAG1769 . DMSO 2 3 1 OT3-65TCCTCGTGTGTGTGTGCGTGTGG1770 . 6CGGGCGGGTGGACAGATGAG1771.AGGAGGTC TCGAGCCA GGGG1772. DMSO 2 3 1GTCTATAT0T3-66TTAAGGTEGGTGTGTGCGTGGGG1773 . 6TCAACCTAGTGAACACAGACCACTGA1774 .ACAGCCCA CAACCTCA1775 . DMSO 1 2 3TGTTGTCATTTOT3-67TTATATTGTGTG TGTGCGTGGGG1776. 6GCCAGGGCCAGTGGATTGCT1777 .CTTAGTATGTCAGCCG1778. DMSO 2 4 0GAOT3-68TTGAGGAGAGTG TGTGCGTGAGG1779 . 6GAGCCCCACCGGTTCAGTCC1780 .GCCAGAGC TACCCACT CGCC1781. DMSO 121782. 1783 , 1784.Target 4GAGTCCGAGCAGAAGAAGAAGGG1785. 0GGAGCAGCTGGTCAGAGGGG1786 .GGGAAGGG GGACACTG GGGA1787 . DMSOATCTGCAC014-1GAGTTAGAGCAGAAGAAGAAAGG1788. 2TCTCTCCTTCAACTCATGACCAGCT1789.ATGTATGTACAGGAGT1790. DMSO 0 1 1CAT0T4-2AAGTCAGAGGAG AAGAAGAAGGG1791. 3AAGACAGAGGAGAA TGGGGMTCTCCAGAAGAAGGG (SEQID NO:2229) AAGAACCCCC1792 .AGGGTGTA CTGTGGGA ACTTTGCA1793 . DMSO 2 1 1ACTTCGTAOT4-3AAGTCCGAGGAG AGGAAGAAAGG1794. 3GATGGCCCCACTGAGCACGT1795.GAGCCTTAAACATGTG1796 . DMSO 1 0 2GC OT4-4AAGTCTGAGCACAAGAAGAATGG1797 .AGGAT TAAT GT TT AAAGTCACTGGTG G1798 ,TCAAACAAGGTGCAGA TACAGCA1799 .IM beta! ne,TD0 2 TGCTCTGT014-5ACGTCTGAGCAGAAGAAGAATGG1800. 3TCCAAGCCACTGG TTTCTCAGTCA1801,GGATCATATTTTGGGG1802 . DMSO 0 1 2GA W O 2014/144592 PC T/U S2014/029068 TABLE E OT4-6GACTCCTAGCAAAAGAAGAATGG1803. 3ACTTTCAGAGCTTGGGGCAGGT1804 ,CCCACGCTGAAGTGCAATGGC1805 . DMSO 1 1 1 OT4-7GAGACTGAGAAGAAGAAGAAAGG1806. 3CAAAGCATGCCTTTCAGCCG1801 ,GGCTCTTCGATTTGGCACCT1808 .IM betai ne,TD1 1 OT4-8GAGCCGGAGCAG AAGAAGGAGGG1809. 3 Nat optimized 1 0 2 OT4-9GAGCCTGAGCAGAAGGAGAAGGG1810. 3GGACTCCCTGCAG CTCCA.GC1811 ,AGGAACACAGGCCAGGCTGG1812 .72CAnne a1, 6% DMSO0 3 0T4-10GAGGCCGAGCAG AAGAAAGACGG1813. 3CCCTTTAGGCACCTTCCCCA1814 ,CCGACCTTCATCCCTCCTGG1815 . DMSO 0 1 2 OT4-11GAGTAAGAGAAG AAGAAGAAGGG1816. 3TGATTCTGCCTTAGAGTCCCAGGT1817 .TGGGCTCTGTGTCCCTACCCA1818 . DMSO 0 3 0f 8sOT4-12GAGTAGGAGGAG AAGAAGAAAGG1819. 3 Not optimized 2 1 0 OT4-13GAGTCCGGGAAG GAGAAGAAAGG1820. 3AGGCAGGAGAGCAAGCAGGT1821 ,ACCCTGAC TACTGACT GACCGCT1822 . DMSO 0 1 2 OT4-14GATTCCTACCAGAAGAAGAATGG1823. 3CTCCCCATTGCGACCCGAGG1824 ,AGAGGCAT TGACTTGG AGCACCT1825 . DMSO 1 2 0 0T4-15GCGACAGAGCAGAAGAAGAAGGG1826. 3CTGGAGCCCAGCAGGAAGGC1827 .CCTCAGGG AGGGGGCC TGAT1828 . DMSO 1 2 0 OT4-16AAATCCAACCAG AAGAAGAAAGG1829. 4ACTGTGGGCGTTG TCCCCAC1830 ,AGGTCGGT GCAGGGTT TAAGGA1831. DMSO 1 0 3 OT4-17AAGTCTGAGGACAAGAAGAATGG1832. 4GGCGCTCCCTTTT TCCCTTTGT1833 ,CGTCACCCATCGTCTC GTGGA1834 . DMSO 2 0 2GCATCTTGOT4-18AAGTTGGAGCAGGAGAAGAAGGG1835. 4TGCCATCTATAGCAGCCCCCT1836 ,CTAACCGTACTTCTTC1837 . DMSO 1 0 3TGA W O 2014/144592 PCT/US2014/029068 TABLE E OT4-19AATACAGAGCAGAAGAAGAATGG1838. 4GTGGAGACGCTAAACCTGTGAGGT1839 .GCTCCTGGCCTCTTCC TACAGC1840 . DMSO 1 2 1 OT4-20AGGTACTAGCAGAAGAAGAAAGG1841. 4CCGAACTTCTGCTGAGCTTGATGC1842 .CCAAGTCAATGGGCAACAAGGGA1843 . DMSO 0 2 2 OT4-21AGGTGCTAGCAG AAGAAGAAGGG1844. 4 Net optimized 1 1 2 0T4-22ASG1GGGAGCAG AAGAAGAAGGG1845. 4TGCCCCCAAGACCTTTCTCC1846 .ATGGCAGG CAGAGGAG GAAG1847 . DMSO 2 0 2 OT4-23CAAACGGAGCAGAAGAAGAAAGG1848. 4GGGTGGGGCCATTGTGGGTT1849 .CTGGGGCCAGGGTTTCTGCC1850 . DMSO 3 0 1 OT4-24CACTCTGAGGAGAAGAAGAAAGG1851. 4TGGAGAACATGAGAGGCTTGCAA1852 .TCCTTCTGTAGGCAATGGGAACAA1853 . DMSO 3 0 1 0T4-25CAGTCATGGCAGAAGAAGAAAGG1854. 4GCCACATGGTAGAAGTCGGC1855 ,GGCAGATTTCCCCCATGCTG1856.IM beta!ne, TD2 1 OT4-26CCGTCCCAGCAG TAGAAGAATGG1857. 4TGTACACCCCAAG TCCTCCC1858 .AAGGGGAG TGTGCAAG COTO1859 . DMSO 3 1 0 OT4-27GTCTGCGATCAGAAGAAGAAAGG1860. 4AGGTCTGGCTAGA GATGCAGCA1861 ,AGTCCAACACTCAGGTGAGACCCT1862 . DMSO 3 1 0GGGTATGGOT4-28TAATCCAATCAGAAGAAGAAGGG1863. 4CCAAGAGGACCCAGCTGTTGGA1864 ,AATTCTGGATTAGCAG1865 . DMSO 0 2 2AGO014-29TATACGGAGCAGAAGAAGAATGG1866. 4ACCATCTCTTCATTGATGAGTCCCAA1867 .ACACTGTGAGTATGCT TGGCGT1868 . DMSO 2 2 0 014-30ACTTCCCTGCAGAAGAAGAAAGG1869. 5GGCTGCGGGGAGATGAGCTC1870 .TCGGATGCTTTTCCAC AGGGCT1871 . DMSO 2 2 1 OT4-31AGGACTGGGCAG AAGAAGAAGGG1872. 5TOT T CCAGGAGGGCAGCTCC1873 ,CCAATCCTGAGCTCCTACAAGGCT1874 . DMSO 1 0 4 W O 2014/144592 PC T/U S2014/029068 TABLE E OT4-32AGGTTGGAGAAGAAGAAGAAGGG1875. 5GAGCTGCACTGGATGGCACT1876 .TGCTGGTT AAGGGGTG TTTTGGA1877 . DMSO 1 1 3 OT4-33AGTTCAGAGCAGGAGAAGAATGG1878. 5TCTGGGAAGGTGAGGAGGCCA1879 .TGGGGGACAATGGAAA AGCAATGA1880 . DMSO 0 2 04-34ATGACACAGCAGAAGAAGAAGGG1881. 5CTTGCTCCCAGCCTGACCCC1882 .AGCCCTTGCCATGCAGGACC1883 . DMSO 3 1 1 OT4-35ATGACAGAGAAG AAGAAGAAAGG1884.GGGATTITTATCT GTTGGGTGCGAA1885 .AACCACAG ATGTACCC TCAAAGCT1886. DMSO 2 2 1 OT4-36CCGCCCCTGCAGAAGAAGAACGG1887.cACCCATCAGGACCGCAGCAC1688 .TCTGGAAC CTGGGAGG CGGA1889 .7Annea 1, 3% DMSO1 1 OT4-37GCAGGAGAGCAGAAGAAGAAAGG1890.CGTCCCTCACAGCCAGCCTC1891 .CCTCCTTG GGCCTGGG GTTC1892 . DMSO 1 3 1 0T4-38GTTCAAGAGCAGAAGAAGAATGG1893.CCCTCTGCAAGGTGGAGTCTCC1894 .AGATGTTCTGTCCCCAGGCCT1895 . DMSO 1 3 1 OT4-39GTTTTGAAGCAGAAGAAGAAAGG1896. 5GGCTTCCACTGCTGAAGGCCT1897 .TGCCGCTCCACATACCCTCC1898 . DMSO 2 1 2AGCACCTAOT4-40TATGGCAAGCAGAAGAAGAAAGG1899. 5AGCATTGCCIGTCGGGTGATGT1900 ,TTGGACACTGGTTGTC1901. DMSO 1 3 1T014-41TGGTGGGATCAG AAGAAGAAAGG1902. 5TCTAGAGCAGGGGCACAATGC1903 .TGGAGATGGA.GCCTGG TGGGA1904 . DMSO 2 2 1 OT4-42ACCCACGGGCAGAAGAAGAAGGG1905. 6GGTCTCAGAAAATGGAGAGAAAGCAC G1906 ,CCCACAGAAACCTGGGCCCT1907 . DMSO 1 2 OT4-43ACTCCTGATCAGAAGAAGAAGGG1908. 6GGTTGCTGATACCAAAACGTTTGCCT1909 .TGGGTCCTCTCCACCTCTGCA1910 . DMSO 0 3 3ACTCTCCTTAA.GT CAGAATCT0T4-44AAGAAGAAAGG1911. 6 ACTGATATGGCTGT1912 . TGCTCTGT TGCCCA1913 . DMSO 0 4 2 W O 2014/144592 PCT/US2014/029068 TABLE E 0T4-45ATTTTAGTGCAGAAGAAGAAAGG1914. 6 Not optimized 2 2 2OT4-46ATITTAGTGCAGAAGAAGAAAGG1915. 6 Not optimized 2 2 2TCCCAAGA0T4-47CCATGGCAGCAGAAGAAGAAGGG1916. 6CAATGCCTGCAGTCCTCAGGA1917 ,GAAAACTOTGTCCTGA1918 . DMSO 4 1 1CA014-48CCATTACAGCAGAAGAAGAAGGG1919. 6GCATTGGCTGCCCAGGGAAA1920 .TGGCTGTGCTGGGCTGTGTT1921 . DMSO 2 2 2 OT4-49CGAGGCGGGCAGAAGAAGAAAGG1922. 6CCACAAGCCTCAGCCTACCCG1923 .ACAGGTGCCAAAACAC TGCCT1924 . DMSO 2 1 3TCATTGCAGCAGAAOT4-50TCATTGCAGCAGAAGAAGAAAGG1925. 6GAAGAAAGG TCATTGTAGCAGAA GAAGAAAGG (SEQGCCl'CTTGCAAAT GAGACTCCTTTT1926.CGAICAGT CCCCTGGC GTCC1927 . DMSO/2/3 2ID NO:2230)OT4-51TCTCCAGGGCAGAAGAAGAAAGG1928. 6TOCCAGAATCT GO CTCCGCA192 9 .AGGGGTTT CCAGGCAC ATGGG1930 . DMSO 0 4 21931. 1932 , 1933 .

Target 5GTCATCTTAGTCATTACCTGAGG1934. 0TCCTAAAAATCAGTTTTGAGATTTACTTCC1935 .AAAGTGTT AGCCAACA TACAGAAG TCAGGA1936 . DMSOGGTATC TAAGTCATTACCTGTGG (SEQ TGTCTGAG0T5-1GGTATCTAAGTC ATTACCTGTGG1937.ID NO:2231)GGTATCTAAGTCAAACATCTGGGGAAAGCAAAAGTCAACA.1938 ,TATCTAGGCTAAAAGT1939 . DMSO/ 1 1TACCTGTGG (SEQ GGTID NO:2232)AGTGCTTT0T5-2GTAATATTAGTCATTACCGGTGG1940. 3ACGATCTTGCTTCATTTCCCTGTACA1941 ,GTGAACTGAAAAGCAA1942 . DMSO 0 3 0ACAOT5-3GTAATCTGAGTC ATTTCCTGGGG1943. 3GCACCTTGGTGCT GCTAAATGCC1944 .GGGCAACTGAACAGGC ATGAATGG1945 . DMSO 1 2 0 W O 2014/144592 PCT/US2014/029068 TABLE E OT5-4GTCATCCTAGTCATTTACTGGGG1946.AACTGTCCTGCATCCCCGCC1947 ,GGTGCACC TGGATCCA CCCA1948 . DM3O 1 1 1 OT5-5GTCATCCTAGTC CTTACCTGAGG1949. 3 Not optimized 1950 . 1951. 1 1 1 0T5-6GTCATCTGAGGC ATTAACTGGGG1952. 3CATCACCCTCCACCAGGCCC1953 ,ACCACTGC TGCAGGCT CCAG1954 .72C Anne a 1, 3% DMSO3 0 0T5-7AATATGTTAGTCATTACCTGAGG1955. 4 Not optimized 2 0 2 OT5-8ATAAACGTAGTC ATTACCTGGGG1956. 4CCTGACCCGTGGT TCCCGAC1957 ,TGGTGCGT GGTGTGTG TGGT1958 .72C Anne a 1, 3% DMSO2 1 0T5-9ATCATCATCGTCATTATCTGGGG1959. 4TGGGAACATTGGAGAAGTTTCCTGA1960 ,CCATGTGACTACTGGGCTGCCC1961. DMSO 1 1 2GGTTCTCT0T5-10ATCATTTTACTCATTACTIGTGG1962. 4AGCCTTGGCAAGCAACTCCCT1963 ,CTCTCAGAAAAGAAAG1964 . DMSO 1 0 3AGGOT5-11ATCATTTTAGTCATCTCCTGTGG1965. 4GECAGCGGACTTCAGAGCCA1966 .GCCAGAGGCTCTCAGCAGTGC1967 . DMSO 1 0 3 0T5-12CACAGCTTAGTC ATCACCTGGGG1968. 4CCAGCCTGGTCAATATGGCA1969 ,ACTGTGCC CAGCCCCA TATT1970 . DMSO 2 1 1 OT5-13CCCAGCTTAGTC ATTAGCTGTGG1971. 4ATGCCAACACTCG AGGGGCC1972 .CGGGTTGTGGCACCGGGTTA1973 . DMSO1 1AGAGTTCAOT5-14CTCACCITTGTC ATTTCCTGAGG1974. 4TTGCTCTAGTGGGGAGGGGG1975 ,GGCATGAA AAGAAGCA1976 . DMSO 3 0 1ACAOT5-15CTCATTTTATTC ATTGCCTGGGG1977. 4AGCTGAAGATAGCAGTGTTTAAGCCT1978 ,TGCAATTT GAGGGGCT CTCTTCA1979 . DMSO 1 1 2 W O 2014/144592 PCT/US2014/029068 TABLE E 16 ־ 015CTCTCCTTAGTC ACTACCTGAGG1980. 4AGTCACTGGAGTAAGCCTGCCT1981 ,TGCCAGCC AAAAGTTG TTAGTGTG T1982 . DMSO 2 0 2 075-17CTTATCTCTGTCATTACCTGGGG1983. 4GGGTCTCCCTCAGTGCCCTG1984 .TGTGTGGT AGGGAGCA AAACGACA1985 . DMSO 2 0 2 OT5-18GACAGCTCCGTCATTACCTGGGG1986. 4TGGGGGCTGTTAAGAGGCACA1987 ,TGACCACA CACACCCC CACG1988 . DMSO 1 2 1 015-19GCCACCTCAGTCATTAGCTGGGG1989. 4TCAAAACAGATTGACCAAGGCCAAAT1990 ,TGTGTTTT TAAGCTGC ACCCCAGG1991. DMSO 1 0 OT5-20GGAATCTTACTCATTACTTGGGG1992. 4TCTGGCACCAGGAC1GATTGTACA1993 .GCACGCAGCTGACTCCCAGA1994 . DMSO 1 2 1 OT5-21GTGGCCICAGTCATTACCTGGGG1995. 4 Net optimized 1 0 3 OT5-22GTTGTTTTAGTG ATTACCTGAGG1996. 4AGCATCTGTGATACCCTACCTGTCT1997 ,ACCAGGGC TGCCACAG AGTC1938 . DMSO 1 0 3 OT5-23TACATCTTAGTCCTCACCTGTGG1999. 4TAGTCTTGTTGCCCAGGCTG2000 .CTCGGCCC CTGAGAGT TCAT2001. DMSO 1 2 1TCCATCTCACTCAT 24 ־ 015TCCATCTCACTCATTACCTGAGG2002. 4TACCTGAGG {SEQ ID NO12233) TCCATCTCACTCATCIGCAACCAGGGC CCTTACC2003 ,GAGCAGCAGCAAAGCC 2004 . DMSO 1 1 2TACCTGAIG (SEQID N0:2234)OT5-25TTCATCCTAGTCAACACCTGGGG2005. 4GCC T GGAGAGCAA GCCTGGG2006.AGCCGAGA CAATCTGC CCCG2007 . DMSO 1 1 2 OT5-26TTTATATTAGTGATTACCTGTGG2008. 4TTTATATTAGTGATTACCTGCGG (SEQID NO:2235)AGTGAAACAAACAAGCAGCAGTCTGA2009 .GGCAGGTC TGACCAGT GGGG2010 .NoDMSOTD2 1TGAGTAGAOT5-27AACGTGIAAGTCATTACCTGAGG2011.AGGCTCAGAGAGGTAAGCAATGGA2012 .CAGAAATG TTACCGGT2013 . DMSO 3 0 2GTT W O 2014/144592 PCT/US2014/029068 TABLE E OT5-28AAGATCACAGTC ATTACCTGGGG2014. 5TCAGAGATGTTAAAGCCTTGGTGGG2015.AGTGAACCAAGGGAATGGGGGA2016 . DMSO 3 0 2 OT5-29AGAATATTAGTC CTTACCTGGGG2017. 5TGTGCTTTCTGGG GTAGTGGCA2018 ,CACCTCAGCCCTGTAG TCCTGG2019 . DMSO 0 4 1 0T5-30AGCAGATTAGTGATTACCTGGGG2020. 5CCATTGGGTGACTGAATGCACA2021,GCCACTGT CCCCAGCC TATT2022 .IM beta! ne, TD3 1 0T5-31AGIAGCTTAGTGATTACCTGGGG2023. 5ACCAAGAAAGT GA AAAGGAAACCC2024 .TGAGATGG CATACGAT TTACCCA2025 . DMSO 1 2 2TGGCATCAOT5-32CACGGCTTACTCATTACCTGGGG2026. 5AGGGTGGGGACTG AAAGGAGCT2027 .CTCAGAGATTGGAACA2028 . DMSO 3 1 1CA025-33CATATGTTAGGC ATTACCTGGGG2029. 5ACCAGTGCTGTGTGACCTTGGA2030.TCCTATGGGAGGGGAGGCTTCT2031. DMSO 3 1 1 OT5-34CATTTCTTAGTCATTTCCTGAGG2032. 5CCAGGTGTGGTGG TTCATGAC2033 .GCATACGGCAGTAGAA TGAGCC2034 .68C, 3%DMSO0 1 oT5-35TGCAGCTAACTC ATTACCTGGGG2035. 5CAGGCGCTGGGTTCTTAGCCT2036 .CCTTCCTG GGCCCCAT GGTG2037 . DMSO 2 3 0 OT5-36TTGCTTITAGTTATTACCTGGGG2038. 5TGGGGTCCAAGATGTCCCCI2039 .TGAAACTGCTTGATGAGGTGTGGA2040 . DMSO 1 2 2ACTTGCAAOT5-37AACTTGAAAGTCATTACCTGTGG2041. 6GCTGGGCTTGGTGGTATATGC2042 .AGCTGATAACTGACTG2043 . DMSO 5 0 1A0T5-38AAGGTCACAGTCATTACCTGGGG044. 6AGTTGGTGTCACTGACAATGGGA2045 .CGCAGCGCACGAGTTCATCA2046. DMSO 3 0 3 015-39AATGTCTTCATCATTACCTGAGG2047. 6AGAGGAGGCACAATTCAACCCCT2048 .GGCTGGGG AGGCCTCA CAAT2049 . DMSO 1 1 4 W O 2014/144592 PCT/US2014/029068 TABLE E 0T5-40AGATGCTTGGTCATTACCTGTGG2050. 6GGGAAAGTTTGGGAAAGTCAGCA2051 ,AGGACAAG CTACCCCA CACC2052 . DMSO 1 3 2 0T5-41AGTAGMTAGTTATTACCTGGGG2053. 6T GG7GCATCAAAG GGTTGCTTCT2054 .TCATTCCA GCACGCCG GGAG2055 . DMSO 0 3TGGAGTAA0T5-42AGTAGGTTAGTAATTACCTGGGG2056. 6CCCAGGCTGCCCATCACACT2057 ,GTATACCTTGGGGACC2058 . DMSO 1 3 2T0T5-43CAAATGAGAGTCATTACCTGAGG2059. 6TCAGTGCCCCTGG GTCCTCA2060 ,TGTGCAAA TACCTAGC ACGGTGC2061 . DMSO 4 2 0ACTGAAGT0T5-44CATGTCTGAATC ATTACCTGAGG2062. 6AGCACTCCCTTTTGAATITTGGTGCT2063 .CCAGCCTCTTCCATTT2064 . DMSO 2 1 3CAOT5-45CCTGACTTGGTCATTACCTGTGG2065. 6GAAACCGGTCCCTGGTUCCA2066 ,GGGGAGTA GAGGGTAG TGTTGCC2067 . DMSO 2 0 4 0T5-46CGTGCATTAGTCATTACCTGAGG2068. 6TIGCGGGTCCCTGTGGAGTC2069 ,AGGTGCCG TGTTGTGC CCAA2070 . DMSO 1 2 3 Target 6GGAATCCCTTCTGCAGCACCTGG2071. 0GCCCTACATCTGCTCTCCCTCCA2072 .GGGCCGGGAAAGAGTTGCTG2073 . DMSO OT6-1GGAACCCCGTCTGCAGCACCAGG2074 . 2TTGGAGTGTGGCC CGGGTTG2075 ,ACCTCTCT TTCTCTGC CTCACTGT2076. DMSO 0 1 1 OT6-2GGAACACCTTCT GCAGCTCCAGG077.CACACCATGCTGATCCA.GGC2078 ,GCAGTACG GAAGCACG AAGC2079 . DMSO 1 1 1 oT6-3GGAAGCTCTGCTGCAGCACCTGG2080. 3CTCCAGGGCTCGCTGTCCAC2081,CTGGGCTC TGCTGGTT CCCC2082 . DMSO 0 2 1 OT6-4GGAATATCTTCT GCAGCCCCAGG2083. 3CTGTGGTAGCCGTGGCCAGG2084 .CCCCATACCACCTCTCCGGGA2085. DMSO 0 2 1 W O 2014/144592 PC T/U S2014/029068 102 TABLE E OT65־GGAATCACTTTTACAGCACCAGG2086. 3GGTGGCGGGACTTGAATGAG2087 ,CCAGCGTGTTTCCAAGGGAT2088 .IM beta! ne,TD1 2 GGAATCCCCTCTCC 0T6-6GGAATCCCCTCT CCAGCCCCTGG2089. 3AGCCCCTGG (SEQ ID NO:2236) GGAATCCCCTCTCC AGCCTCTGG(SEQCCAGAGGTGGGGCCCTGTGA2090 .TTTCCACA 'CTCAGTTCTGCAGGA2091. DMSO 1 1 1/2 ID NO:2237)0T6-7GGAATCTCTTCTTCAGCATCTGG2092 .GGAATCTCTTCCTT GGCATCTGG(SEQ ID NO:2238)TGTGACTGGTTGTCCTGCTTTCCT2093 ,GCAGTGTTTTGTGGTGATGGGCA2094 .IM beta!ne, TD1 5 OT6-8GGAATTGCTTCT GCAGCGCCAGG2095. 3CTGGCCAAGGGGTGAGTGGG2096.TGGGACCC CAGCAGCC AATG2097 . DMSO 1 0 2 016-9GGACTCCCCTCT GCAGCAGCTGG2098. 3ACGGTGTGCTGGCTGCTCTT2099 ,ACAGTGCTGACCGTGC TGGG2100 . DMSO 1 1 1 e u OT6-10GGAGTCCCTCCTACAGCACCAGG2101. 3TGGTTTGGGCCTCAGGGATGG2102 ,TGCCTCCC ACAAAAAT GTCTACCT2103 . DMSO 0 0 3 016-11GGAGTCCCTCCTACAGCACCAGG2104.TGGTTTGGGCCTCAGGGATGG2105 .ACCCCTTATCCCAGAACCCATGA2106 . DMSO 0 0 3 12 ־ 016GGGATCCATTCTCCAGCCCCTGG2107.TCCAAGTCAGCGATGAGGGCT2108 .TGGGAGCTGTTCCTTT TTGGCCA2109 . DMSO 0 3 0 OT6-13GGCTTCCCTTCTGCAGCGCCAGG2110. 3CACCCCTCTCAGCTTCCCAA2111 .GCTAGAGGGTCTGCTGCCTT2112 . DMSO 1 2 0 OT6-14TGAATCCCATCTCCAGCACCAGG2113L 3AGACCCCTTGGCCAAGCACA.2114 .CTTGCTCT CACCCCGC CTCC2115 . DMSO 2 1 0TCTCACTT016-15AAAATACCTTCT GCAGTACCAGG2116. 4ACATGTGGGAGGCGGACAGA2117 ,TGCIGTTACCGATGTC G2118 . DMSO 0 1 3 W O 2014/144592 PCT/US2014/029068 TABLE E 0T6-16AAAATCCCTTCT TCAACACCTGG2119. 4GGA.CGACTGTGCCTGGGACA2120 ,AGTGCCCAGAGTGTTGTAACTGCT2121.72C Annea 1, 3% DMSO1 3 OT6-17ACACTCCCTCCTGCAGCACCTGG2122. 4GGAGAGCTCAGCGCCAGGTC2123 ,CAGCGTGG CCCGTGGG AATA2124 . DMSO 1 1 2ACCCCACT־ 016ACCATCCCTCCT GCAGCACCAGG2125. 4GCTGAAGTGCTCTGGGGTGCT2126 ,GTGGATGAATTGGTAC2127 . DMSO 1 1 2COT6-19AGAGGCCCCTCTGCAGCACCAGG2128. 4TCGGGGTGCACATGGCCATC2129 .TTGCCTCG CAGGGGAA GCAG2130 . DMSO 0 1 3 OT6-20AGGATCCCTTGTGCAGCTCCTGG2131. 4CTCGTGGGAGGCCAACACCT2132 .AGCCACCAACACATAC CAGGCT2133 . DMSO 2 0 2 oT6-21CCACTCCTTTCTGCAGCACCCGG2134. 4GCATGCCTTTAATCCCGGCT2135 ,AGGATTTC AGAGTGAT GGGGCT2136 . DMSO 2 1 1GCAAATTTOT6-22GAAGGCCCTTCAGCAGCACCTGG2137. 4CGCCCAGCCACAAAGTGCAT2138 ,CTGCACCTACTCTAGG2139 . DMSO 1 1 2CCT016-23GATATCCCTTCTGTATCACCTGG2140. 4AGCTCACAAGAATTGGAGGTAACAGT2141 ,GCAGTCACCCTTCACT GCCTGT2142. DMSO 1 1 2 OT6-24GGGTCCGCTTCTGCAGCACCTGG2143. 4AAACIGGGCTGGGCTTCCGG2144 .GGGGCTAAGGCATTGT CAGACCC2145 . DMSO 2 0 2 ־ 016GTCTCCCCTTCTGCAGCACCAGG2146. 4GCAGGTAGGCAGTCTGGGGC2147 ,TCTCCTGCCTCAGCCTCCCA214a .IM betai ne,TD2 1 016-26GTCTCCCCTTCTGCAGCACCAGG2149. 4GCAGGTAGGCAGT CTGGGGC2150 .TCTCCTGC CTCAGCCT CCCA2151.IM betai ne,TD2 1 W O 2014/144592 PC T/U S2014/029068 104 TABLE E OT6-27GTCTCCCCTTCTGCAGCACCAGG2152. 4GCAGGTA-GGCAGTCTGGGGC2153.TCTCCTGCCTCAGCCICCCA2154 .IM beta! ne, ID2 1 OT6-28TCATTCCCGTCT GCAGCACCCGG2155. 4GCTCTGGGGTAGAAGGAGGC2156.GGCCTGTCAACCAACCAACC2157 . DMSO 2 2 0 016-29TGCACCCCTCCTGCAGCACCAGG2158. 4TGACATGTTGTGTGCTGGGC2159,AAATCCTGCAGCCTCCCC1T2160. DMSO 0 2 2 ־ 016TGCATACCCTCTGCAGCACCAGG2161. 4TCCTGGTGAGATC GTCCACAGGA2162 ,TCCICCCCACTCAGCCTCCC2163. DMSO 0 3 1 016-31TGCATGGCTTCT GCAGCAC C AG G2164. 4TCCTAATCCAAGTCCITTGTTCAGACA2165 .AGGGACCAGCCAC1ACCCI1CA2166 . DMSO 2 2 0 016-32AATATTCCCTCTGCAGCACCAGG2167.GGGACACCAG1TCCTTCCAT2168 .GGGGGAGATTGGAGTT cccc2169 . DMSO 1 0 4 016-33ACCATTTC1ICTGCAGCACCTGG2170.GACACCACTATCAAGGCAGAGTAGGT2171 ,TCTGCCTG GGGIGC1I 1CCC2172 . DMSO 1 1 3 OT6-34AGCTCCCATTCTGCAGCACCCGG2173. 5CIGG G AijC GGGAAGTGC2174 ,GCCCCGAC AGATGAGG CCTC2175 . DMSO 1 2 2 016-35CAGATTCCTGCTGCAGCACCGGG2176. 5CAGATTACTGCTGCAGCACCGGG (SEQ ID NO:2239)CGGGTCTCGGAATGCCTCCA2177 .ACCCAGGAA1TGCCACCCCC2178 . DMSO 1 2 3 016-36CCAAGAGCTTCT GCAGCACCTGG2179. 5TTGCTGTGGTCCCGG1GG1G2180 .GCAGACAC TAGAGCCC GCCC2181. DMSO 3 2 0 016-37CCCAGCCCTGCTGCAGCACCCGG2182.GGTGTGGTGACAGG1CGGGI2183 .ACCTGCGTCTCTGTGC1GCA2184 . DMSO 2 3 0 016-38CCCCTCCCTCCTGCAGCACCCGG2185.CTCCCAGGACAGT GCTCGGC2186 ,CC1GGCCC CATGCTGC CIG2187 . DMSO 2 2 1 016-39CTACTGACTTCT GCAGCACCTGG2188.c;TGCGTAGGT1TTGCCTCTGTGA2189 .AGGGAATGATGTTT1CCACCCCC12190 . DMSO 2 3 0 W O 2014/144592 PC T/U S2014/029068 TABLE E OT6-40CTCCTCCCTCCTGCAGCACCTGG2191. 5CTCCGCAGCCACCGTTGGTA2192 ,TGCATTGACGTAC3ATGGCTCA2193 . DMSO 1 3 1 OT6-41TCTGTCCCTCCTGCAGCACCTGG2194.ACCTGCAGCATGAACTCTCGCA2195.ACCTGAGCAACATGACTCACCTGG2196 . DMSC 2 1 2ACACAAACTT CTGC־ 076ACACAAACTTCTGCAGCACCTGG2197. 6AGCACCTGGACACAAACTTCTGCAGCACGTGG(SEQTCTCCAGTTTCTTGCTCTCATGG2198 .ACCATTGGTGAACCCAGICA2199 .betai ne,/3 1ID NO:2240)TCAGCTATOT6-43ACTGTCATTTCTGCAGCACCTGG2200. 6TGGGGTGGTGGTC TTGAATCCA2201 ,AACCTGGGACTTGTGC2202 . DMSO 2 1 3TOT6--44ACTTTATCTTCTGCAGCACCTGG2203. 6AGCAGCCAGTCCAGTGTCCTG2204 ,CCCTTTCA TCGAGAAC CCCAGGG2205 . DMSO 3 1 2 OT6-45ATCCTTTCTTCTGCAGCACCTGG2206. 6TGGACGCTGCTGGGAGGAGA2207 .GAGGTCTCGGGCTGCTCGTG2208 . DMSO 0 3 3 OT6-46CACCACCGTTCTGCAGCACCAGG2209. 6AGGTTTGCACTCTGTTGCCTGG2210.7GGGGTGA7TGGTTGCCAGGT2211. DMSO 3 2 17GCAGGAA0T6-47CATGTGGCTTCTGCAGCACCTGG2212. 6TCTTCCTTTGCCAGGCAGCACA2213 .TAGCAGGTATGAGGAG2214 , DMSO 4 0 2T0T6-48CATTTTCTTTCTGCAGCACCTGG2215. 6GGACGCCTACTGCCTGGACC2216,GCCCTGGCAGCCCATGGTAC2217 . DMSO 3 0 3 OT6-49CTCTGTCCTICTGCAGCACCTGG2218. 6AGGCAGTCATCGCCTTGCTA2219 ,GGTCCCACCTTCCCCTACAA2220 . DMSO 21 OT6-50CrGTACCCTCCTGCAGCACCAGG2221. 6 Not optimized 3 1 2 51 ־ 076TTGAGGCCGTCT GCAGCACCGGG2222. 6CCCCAGCCCCCACCAGTITC2223 .CAGCCCAG GCCACAGC TTCA2224 . DMSO 1 4 1 W O 2014/144592 PCT/US2014/029068 PCT/US2014/029068 WO 2014/144592 107 Sanger sequencing for quantifying frequencies of indel mutations Purified PCR products used for T7EI assay were ligated into a Zero Blunt TOPO vector (Life Technologies) and transformed into chemically competent Top bacterial cells. Plasmid DNAs were isolated and sequenced by the Massachusetts General Hospital (MGH) DNA Automation Core, using anM13 forward primer (5’- GTAAAACGACGGCCAG-3‘) (SEQ IDNO:1059). Restriction digest assay for quantifying specific alterations induced by HDRwith ssODNs PCR reactions of specific 011-target sites were performed using Phusion high- fidelity DNA polymerase (New England Biolabs). The VEGF and EMX1 loci were amplified using a touchdown PCR program ((98 °C, 10 s; 72-62 °C, 1־ °C/cycle, s; 72 °C, 30 s) x 10 cycles, (98 °C, 10 s; 62 °C, 15 s; 72 °C, 30 s) x 25 cycles), with 3% DMSO. The primers used for these PCR reactions are listed in Table E. PCR products were purified by Ampure XP beads (Agencourt) according to the manufacturer ’s instructions. For detection of the BamHY restriction site encoded by the ssODN donor template, 200 ng of purified PCR products were digested with BamHI at 37 °C for 45 minutes. The digested products were purified by Ampure XP beads (Agencourt), eluted in 20ul O.lxEB buffer and analyzed and quantified using a QIAXCEL capillary electrophoresis system. TruSeq library Generation and Sequencing Data Analysis Locus-specific primers were designed to flank on-target and potential and verified off-target sites to produce PCR products ~300bp to 400 bps in length. Genomic DNAs from the pooled duplicate samples described above were used as templates for PCR. All PCR products were purified by Ampure XP beads (Agencourt) per the manufacturer ’s instructions. Purified PCR products were quantified on a QIAXCEL capillary electrophoresis system. PCR products for each locus were amplified from each of the pooled duplicate samples (described above), purified, quantified, and then pooled together in equal quantities for deep sequencing. Pooled amplicons were ligated with dual-indexed Illumina TruSeq adaptors as previously described (Fisher et al., 2011). The libraries were purified and run on a QIAXCEL capillary electrophoresis system to verify change in size following adaptor ligation. The adapter-ligated libraries were quantified by qPCR and then sequenced using Illumina MiSeq 250 bp paired-end reads performed by the Dana-Farber Cancer Institute Molecular Biology Core Facilities. We analyzed between 75,000 and WO 2014/144592 PCT/US2014/029068 108 1,270,000 (average -422,000) reads for each sample. The TruSeq reads were analyzed for rates of indel mutagenesis as previously described (Sander et al., 2013).Specificity ratios were calculated as the ratio of observed mutagenesis at an on-target locus to that of a particular off-target locus as determined by deep sequencing. Fold- improvements in specificity with tru-RGNs for individual off-target sites were calculated as the specificity ratio observed with tru-gRNAs to the specificity ratio for that same target with the matched fall-length gRNA. As mentioned in the text, for some of the off-target sites, no indel mutations were detected with tru-gRNAs. In these cases, we used a Poisson calculator to determine with a 95% confidence that the upper limit of the actual number of mutated sequences would be three in number. We then used this upper bound to estimate the minimum fold-improvement in specificity for these off-target sites.

Example 2a. Truncated gRNAs can efficiently direct Cas9-mediated genome editing in human cells To test the hypothesis that gRNAs truncated at their 5 ’ end might function as efficiently as their full-length counterparts, a series of progressively shorter gRNAs were initially constructed as described above for a single target site in the EGFP reporter gene, with the following sequence: 5’- GGCGAGGGCGATGCCACCTACGG-3‘ (SEQ IDNO:2241). This particular EGFP site was chosen because it was possible to make gRNAs to it with 15, 17, 19, and 20 nts of complementarity that each have a G at their 5’ end (required for efficient expression from the U6 promoter used in these experiments). Using a human cell- based reporter assay in which the frequency of RGN-induced indels could be quantified by assessing disruption of a single integrated and constitutively expressed enhanced green fluorescent protein (EGFP) gene (Example 1 and Fu et al., 2013; Reyon et al., 2012) (Figure 2B),the abilities of these variable-length gRNAs to direct Cas9-induced indels at the target site were measured.As noted above, gRNAs bearing longer lengths of complementarity (21, 23, and 25 nts) exhibit decreased activities relative to the standard full-length gRNA containing 20 nts of complementary sequence (Figure 2H), a result that matches those recently reported by others (Ran et al., Ceil 2013). However, gRNAs bearing 17 or nts of target complementarity showed activities comparable to or higher than the PCT/US2014/029068 WO 2014/144592 109 full-length gRNA, while a shorter gRNA bearing only 15 nts of complementary failed to show significant activity (Figure 2H). To test the generality of these initial findings, full-length gRNAs and matched gRNAs bearing 18, 17 and/or 16 nts of complementarity to four additional EGFP reporter gene sites (EGFP sites #1, #2, #3, and #4; Figure 3A)were assayed. At all four target sites, gRNAs bearing 17 and/or 18 nts of complementarity functioned as efficiently as (or, in one case, more efficiently than) their matched full-length gRNAs to induce Cas9-mediated disruption of EGFP expression (Figure 3A).However, gRNAs with only 16 nts of complementarity showed significantly decreased or undetectable activities on the two sites for which they could be made (Figure 3A). For each of the different sites tested, we transfected the same amounts of the full- length or shortened gRNA expression plasmid and Cas9 expression plasmid. Control experiments in which we varied the amounts of Cas9 and truncated gRNA expression plasmids transfected for EGFP sites #1, #2, and #3 suggested that shortened gRNAs function equivalently to their full-length counterparts (Figures 3E (bottom) and 3F (bottom))and that therefore we could use the same amounts of plasmids when making comparisons at any given target site, Taken together, these results provide evidence that shortened gRNAs bearing 17 or 18 nts of complementarity can generally function as efficiently as full-length gRNAs and hereafter the truncated gRNAs with these complementarity lengths are referred to as "tru-gRNAs" and RGNs using these tru-gRNAs as "tru-RGNs". Whether tru-RGNs could efficiently induce indels on chromatinized endogenous gene targets was tested next, Tru-gRNAs were constructed for seven sites in three endogenous human genes (VEGFA, EMX1, and CLTA including four sites that had previously been targeted with standard full-length gRNAs in three endogenous human genes: VEGFA site 1, VEGFA site 3, EMX1, and CTLA (Example andFu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013) (Figure 3B).(It was not possible to test a tru-gRNA for VEGFA site 2 from Example 1, because this target sequence does not have the G at either position 17 or 18 of the complementarity region required for gRNA expression from a U6 promoter,) Using a well-established T7 Endonuclease I (T7EI) genotyping assay (Reyon et al., 2012) as described above, the Cas9-mediated indel mutation frequencies induced by each of these various gRNAs at their respective target sites was quantified in human U2OS.EGFP cells. For all five of the seven four sites, tru-RGNs robustly induced indel mutations with WO 2014/144592 PCT/US2014/029068 110 efficiencies comparable to those mediated by matched standard RGNs (Figure 3B). For the two sites on which tru-RGNs showed lower activities than their full-length counterparts, we note that the absolute rates of mutagenesis were still high (means of 13.3% and 16.6%) at levels that would be useful for most applications. Sanger sequencing for three of these target sites (VEGFA sites 1 and 3 and EMXE) confirmed that indels induced by tru-RGNs originate at the expected site of cleavage and that these mutations are essentially indistinguishable from those induced with standard RGNs (Figure 3Cand Figures 7A-D). We also found that tru-gRNAs bearing a mismatched 5’ G and an 18 nt complementarity region could efficiently direct Cas9-induced indels whereas those bearing a mismatched 5 ’ G and a 17 nt complementarity region showed lower or undetectable activities compared with matched full-length gRNAs (Figure 7E), consistent with our findings that a minimum of 17 nts of complementarity is required for efficient RGN activity.To farther assess the genome-editing capabilities of tru-RGNs, their abilities to induce precise sequence alterations via HDR with ssODN donor templates were tested. Previous studies have shown that Cas9-induced breaks can stimulate the introduction of sequence from a homologous ssODN donor into an endogenous locus in human cells (Cong et al., 2013; Mali et al., 2013c; Ran et al., 2013; Yang et al., 2013). Therefore, the abilities were compared of matched full-length and tru-gRNAs targeted to VEGFA site 1 and to the EMX1 site to introduce a BamHY restriction site encoded on homologous ssODNs into these endogenous genes. At both sites, tru- RGNs mediated introduction of the BamHY site with efficiencies comparable to those seen with standard RGNs harboring their full-length gRNA counterparts (Figure 3D). Taken together, this data demonstrate that tru-RGNs can function as efficiently as standard RGNs to direct both indels and precise HDR-mediated genome editing events in human cells.

Example 2b. tru-RGNs exhibit enhanced sensitivities to gRNA/DNA interface mismatches Having established that tru-RGNs can function efficiently to induce on-target genome editing alterations, whether these nucleases would show greater sensitivity to mismatches at the gRNA/DNA interface was tested. To assess this, a systematic series of variants was constructed for the tru-gRNAs that were previously tested on PCT/US2014/029068 WO 2014/144592 111 EGFP sites #1, #2, and #3 (Figure 3Aabove). The variant gRNAs harbor single Watson-Crick substitutions at each position within the complementarity region (with the exception of the 5’ G required for expression from the U6 promoter) (Figure5A). The human cell-based EGFP disruption assay was used to assess the relative abilities of these variant tru-gRNAs and an analogous set of matched variant full-length gRNAs made to the same three sites as described in Example 1 to direct Cas9- mediated indels. The results show that for all three EGFP target sites, tru-RGNs generally showed greater sensitivities to single mismatches than standard RGNs harboring matched full-length gRNAs (compare bottom and top panels of Figure5A). The magnitude of sensitivity varied by site, with the greatest differences observed for sites #2 and #3, whose tru-gRNAs harbored 17 nts of complementarity.Encouraged by the increased sensitivity of tru-RGNs to single nucleotide mismatches, we next sought to examine the effects of systematically mismatching two adjacent positions at the gRNA-DNA interface. We therefore made variants of the tru-gRNAs targeted to EGFP target sites #1, #2, and #3, each bearing Watson-Crick transversion substitutions at two adjacent nucleotide positions (Figure 5B).As judged by the EGFP disruption assay, the effects of adjacent double mismatches on RGN activity were again substantially greater for tru-gRNAs than for the analogous variants made in Example 1 for matched full-length gRNAs targeted to all three EGFP target sites (compare bottom to top panels in Figure 5B).These effects appeared to be site-dependent with nearly all of the double-mismatched tru-gRNAs for EGFP sites #2 and #3 failing to show an increase in EGFP disruption activities relative to a control gRNA lacking a complementarity region and with only three of the mismatched tru-gRNA variants for EGFP site #1 showing any residual activities (Figure 5B).In addition, although double mutations generally showed greater effects on the 5 ’ end with full-length gRNAs, this effect was not observed with tru-gRNAs. Taken together, our data suggest that tru-gRNAs exhibit greater sensitivities than full- length gRNAs to single and double Watson-Crick transversion mismatches at the gRNA-DNA interface, Example 2c. tru-RGNs targeted to endogenous genes show improved specificities in human cells The next experiments were performed to determine whether tru-RGNs might show reduced genomic off-target effects in human cells relative to standard RGNs PCT/US2014/029068 WO 2014/144592 112 harboring full-length gRNA counterparts. We examined matched full-length and tru- gRNAs targeted to VEGFA site 1, VEGFA site 3, and EMX1 site 1 (described in Fig. 3B above) because previous studies (see Example 1 and Fu et al., 2013; Hsu et al., 2013) had defined 13 bona fide off-target sites for the full-length gRNAs targeted to these sites. (We were unable to test a tru-gRNA for VEGFA site 2 from our original study6 because this target sequence does not have the G at either position 17 or 18 of the complementarity region required for efficient gRNA expression from a Upromoter.) Strikingly, wc found that tru-RGNs showed substantially reduced mutagenesis activity in human U2OS.EGFP cells relative to matched standard RGNs at all 13 of these bona fide off-target sites as judged by T7EI assay (Table 3 A); for of the 13 off-target sites, the mutation frequency with tru-RGNs dropped below the reliable detection limit of the T7EI assay (2 - 5%) (Table 3A). We observed similar results when these matched pairs of standard and tru-RGNs were tested at the same off-target sites in another human cell line (FT-HEK293 cells) (Table 3A).To quantify the magnitude of specificity improvement observed with tru- RGNs, we measured off-target mutation frequencies using high-throughput sequencing, which provides a more sensitive method for detecting and quantifying low frequency mutations than the T7EI assay, We assessed a subset of 12 of the bona fide off-target sites for which we had seen decreased mutation rates with tru- gRNAs by T7EI assay (for technical reasons, we were unable to amplify the required shorter amplicon for one of the sites) and also examined an additional off-target site for EMX1 site 1 that had been identified by another group? (Fig. 6A). For all 13 off- target sites we tested, tru-RGNs showed substantially decreased absolute frequencies of mutagenesis relative to matched standard RGNs (Fig. 6A and Table 3B) and yielded improvements in specificity of as much as ~5000-fold or more relative to their standard RGN counterparts (Fig. 6B). For two off-target sites (OT1-4 and OT1-11), it was difficult to quantify the on-target to off-target ratios for tru-RGNs because the absolute number and frequency of indel mutations induced by tru-RGNs fell to background or near-background levels. Thus, the ratio of on-target to off-target rates would calculate to be infinite in these cases. To address this, we instead identified the maximum likely indel frequency with a 95% confidence level for these sites and then used this conservative estimate to calculate the minimum likely magnitude of specificity improvement for tru-RGNs relative to standard RGNs for these off-targets.

WO 2014/144592 PCT/US2014/029068 113 These calculations suggest tru-RGNs yield improvements of ~10,000-fold or more at these sites (Fig. 6B).To further explore the specificity of tru-RGNs, we examined their abilities to induce off-target mutations at additional closely related sites in the human genome. For the tru-gRNAS to VEGFA site 1 and EMX1, which each possess 18 nts of target site complementarity, we computationally identified all additional sites in the human genome mismatched at one or two positions within the complementarity region (not already examined above in Table 3A)and a subset of all sites mismatched at three positions among which we favored mismatches in the 5 ’ end of the site as described in Example 1. For the tru-gRNA to VEGFA site 3, which possesses 17 nts of target site complementarity, we identified all sites mismatched at one position and a subset of all sites mismatched at two positions among which mismatches in the 5’ end were favored (again not already examined in Table 3A).This computational analysis yielded a total of 30, 30, and 34 additional potential off-target sites for the tru-RGNs targeted to VEGFA site 1, VEFGA site 3, and the EMX1 site, respectively, which we then assessed for mutations using T7EI assay in human U2OS.EGFP and HEK2cells in which the RGNs had been expressed.Strikingly, the three tru-RGNs to VEGFA site 1, VEFGA site 3, and EMX1 did not induce detectable Cas9-mediated indel mutations at 93 of the 94 potential off- target sites examined in human U2OS.EGFP cells or at any of the 94 potential off- target sites in human HEK293 cells (Table 3C).For the one site at which off-target mutations were seen, whether the standard RGN with a full-length gRNA targeted to VEGFA site 1 could also mutagenize this same off-target site was examined; it induced detectable mutations albeit at a slightly lower frequency (Figure 6C).The lack of improvement observed with shortening of the gRNA at this off-target site can be understood by comparing the 20 and 18 nt sequences for the full-length and tiu- gRNAs, which shows that the two additional bases in the full-length 20 nt target are both mismatched (Figure 6C).In summary, based on this survey of 94 additional potential off-target sites, shortening of the gRNA does not appear to induce new high- frequency off-target mutations.Deep sequencing of a subset of the 30 most closely matched potential off- target sites from this set of 94 site (i.e.—those with one or two mismatches) showed either undetectable or very low rates of indel mutations (Table 3D)comparable to what wc observed at other previously identified off-target sites (Table 3B).We WO 2014/144592 PCT/US2014/029068 114 conclude that tru-RGNs generally appear to induce either very low or undetectable levels of mutations at sites that differ by one or two mismatches from the on-target site. This contrasts with standard RGNs for which it was relatively easy to find high- frequency off-target mutations at sites that differed by as many as five mismatches(see Example 1).

TABLE3Ai On- and off-target mutation frequencies of matched tru-RGNs and standard RGNs ؛; targeted to endogenous genes in human U2OS.EGFP and HEK293 cells ; Target ؛ SEQ * ;20mer Target i!D NO:tIndel mutation frequency (%) ± s.e.m.,؛ TruncatedTarget ;؛ Indel mutation frequency (%). s.e.m ± ؛Gene; IDU2OS.EGFP HEK293U2OS.EGFP HEK293؛ T1 GGGTGGGGGGAGTTTGCTCCtGG ;2242. 23.69 ±1.99 6.98 ±1.33 GTGGGGGGAGTITGCTCCIGG 2243.23.93 ±4.378.34 ±0.01l/EGFA OT1-3 GGATGGAGGGAGTTTGCTCCtGG 2244؛. 17.25 ±2.97 7.26 + 0.62 ATGGAGGGAGTTTGCTCCtGG 2245.N.D. ND. /GDCC3 0T1-4 GGGAGGGTGGAGTTTGCTCCtGG ^2246. 6.23 ± 0.2C 2.66 ±0.30 GAGGGTGGAGTTTGCTCCIGG 22A1. N.D.؛N.D. LOC116437 OT1-6 CGGGGGAGGGAGTTTGCTCCtGG ^2248. ؛3.73 + 0.23 1.41 ±0.07 GGGGAGGGAGTTTGCTCCtGG 2249.N.D. N.D. CACNA2D ־ OT1-11 GGGGAGGGGAAGTTTGCTCCtGG ؛ . 2250 ؛10.4 ±0.7 3.61 ±0.02 GGAGGGGAAGTTTGCTCCtGG 2251.N.D.,. X .״N.D.״״״״,״״״,״״.

T3 GGTGAGTGAGTGTGTGCGTGtGG 2252. 54.08 ± 1.02" 22797 10.17 ؟ GAGTGAGTGTGTGCGTGtGG 2253.50.49 ±1.25" 65± ״ 2O ־"0.01 VEGFA ..5 OT3-1 GGTGAGTGAGTGTGTGTGTGaGG 2254. 6.16 ±0.98 6.02 ±0.11 GAGTGAGTGTGTGTGTGaGG 2255.N.D. N.D. (abParts) .z OT3-2 ; AGTGAGTGAGTGTGTGTGTGgGG 2256. 19.64+ 1.0611.29± ־0.27 ، GAGTGAGTGTGTGTGTGgGG 2257؛.5.52 ±0.253.41 ± 0.07 MAX OT3-4i GCTGAGTGAGTGTATGCGTGIGG 2258־. 7.95 ±0.11 4.50 ± 0.02 GAGTGAGTGTATGCGTGtGG 2259.1.69 + 0.261.27 ± 0.10 OT3-9 GGTGAGTGAGTGCGTGCGGGtGG 2260؛. N.D. 1.09 + 0.17 GAGTGAGTGCGTGCGGGtGG i2261.N.D. N.D. TPCN2 OT3-17 GTTGAGTGAATGTGTGCGTGaGG 2262. 1.85 ±0.08 N.D. GAGTGAATGTGTGCGTGaGG 2263.N.D. N.D. SLIT1 OT3-18 TGTGGGTGAGTGTGTGCGTGaGG £264. 6.16 ±0.56 6.27 + 0.09 GGGTGAGTGTGTGCGTGaGG 2265.N.D. N.D. COMDA OT3-20 AGAGAGTGAGTGTGTGCATGaGGj2266. ؛10.47± 1.08 = 4.38 ± 0.58 GAGTGAGTGTGTGCATGaGG 2267.N.D. N.D. --------- - T4 GAGTCCGAGCAGAAGAAGAAgGG 2268. 41.56 ±0.20] 12.65 ± = 0.31 GTCCGAGCAGAAGAAGAAgGG 2269 ؛. •'V'" 43.01 ± 0.87"7725"+"0.64 •'V* EMX1 A OT4-1 GAGTTAGAGCAGAAGAAGAAaGG 2270. 19.26 + 0.73 4.14 ±0.66 GTTAGAGCAGAAGAAGAAaGG j 2271.N.D.ןN.D. HCN1 = OT-M Hsu31 GAGTCTAAGCAGAAGAAGAAqAG 2272. 4.37 ± 0.58 N.D. ؛ GTCTAAGCAGAAGAAGAAgAG 2273.N.D.N.D. MFAP1 Mutation frequencies were measured by T7EI assay. Means of duplicate measurements are shown with error bars representingstandard errors of the mean. *Off-target site OT4 53 is the same as EMX1 target 3 OT31 from Hsu. et al., 2013.

W O 2014/144592 _ c PCT/US2014/029068 WO 2014/144592 PCT/US2014/029068 116 Table 3BNumbers of wild-type (WT) and indel mutation sequencing readsfrom deep sequencing experiments Site Control tru-RGN Standard RGN Indel WT Freq. Indel WT Freq. Indel WT Freq. VEGFA site 1 45 140169 0.03% 122858 242127 33.66% 150652 410479 26.85% OT1-3 0 132152 0.00% 1595 205878 0.77% 50973 144895 26.02% OT1-4 0 133508 0,00% 0 223881 0.00% 22385 240873 8.50% OT1-6 3 213642 0.00% 339 393124 0.09% 24332 424458 5.21% OT1-11 1 930894 0.00% 0 274779 0.00% 43738 212212 17.09% VEGFA site 3 5 212571 0.00% 303913 292413 50.96% 183626 174740 51.24% OT3-2 1169 162545 0.71% 9415 277616 3.28% 26545 222482 10.66% OT3-4 7 383006 0.00% 15551 1135673 1.35% 42699 546203 7.25% OT3-9 73 145367 0.05% 113 227874 0.05% 1923 168293 1.13% OT3-17 8 460498 0.00% 31 1271276 0,00% 16760 675708 2.42% OT3-18 7 373571 0.00% 284 1275982 0.02% 72354 599030 10.78% OT3-20 5 140848 0.00% 593 325162 0.18% 30486 202733 13.07% EMX1 site 1 1 158838 0.00% 49104 102805 32.32% 128307 307584 29.44% OT4-1 10 169476 0.01% 13 234039 0.01% 47426 125683 27.40% OT4-52 2 75156 0.00% 10 231090 0.00% 429 340201 0.13% OT4-53 0 234069 0.00% 6 367811 0.00% 17421 351667 4.72% Freq. - frequency of indel mutations = number of indel sequences/number of wild-type sequences. Control gRNA = gRNA lacking a complementarity region Table 3CIndel mutation frequencies at potential off-target sites of tru-RGNs targeted to endogenous genes in human cells Target ID Target Site + PAMSEQ ID NO:Number of mismatches Indel mutation frequency (%) ± s.e.m.U2OS.EGFP cellsHEK293 cells145Z9754Site 1GTGGGGGGAGTTTGCTCCtGG2274.(on-target) 23.93 ±4.37 8.34 ±0.01 GTGGGGGGAGTTTGCCCCaGG2275. 1 Not detected Not detectedGTGGGGGGTGTTTGCTCCcGG2276. 1 Not detected Not detectedGTGGGTGGAGTTTGCTACtGG2277. 2 Not detected Not detectedGTGGGGGGAGCTTTCTCCtGG2278. 2 Not detected Not detectedGTGGGTGGCGTTTGCTCCaGG2279. 2 Not detected Not detectedGTGGAGGGAGCTTGCTCCtGG2280. 2 6.88 ± 0.19 Not detectedGTGGGTGGAGTTTGCTACaGG2281. 2 Not detected Not detectedGGGGGGGCAGTTTGCTCCtGG2282. 2 Not detected Not detected PCT/US2014/029068 WO 2014/144592 117 Table 30Indel mutation frequencies at potential off-target sites of tru-RGNs targeted to endogenous genes in human cellsTarget ID Target Site + PAM SEQ IDNumber of mismatchesIndel mutation frequency (%) ± s.e.m. GTGTGGGGAATTTGCTCCaGG 2283. 2 Not detected Not detected CTGCTGGGAGTTTGCTCCtGG 2284. 3 Not detected Not detected TTTGGGAGAGTTTGCTCCaGG 2285. 3 Not detected Not detected CTGAGGGCAGTTTGCTCCaGG 2286. 3 Not detected Not detected GTAAGGGAAGTTTGCTCCtGG 2287. 3 Not detected Not detected GGGGGTAGAGTTTGCTCCaGG 2288. 3 Not detected Not detectedGGGTGGGGACTTTGCTCCaGG2289. 3 Not detected Not detected GGGGGAGCAGTTTGCTCCaGG 2290. 3 Not detected Not detected TTGGGGTTAGTTTGCTCCtGG 2291. 3 Not detected Not detected TTGAGGGGAGTCTGCTCCaGG 2292. 3 Not detected Not detected CTGGGGTGATTTTGCTCCtGG 2293. 3 Not detected Not detected GAGAGGGGAGTTGGCTCCtGG 2294. 3 Not detected Not detectedTTTGGGGGAGTTTGCCCCaGG2295. 3 Not detected Not detectedTTCGGGGGAGTTTGCGCCgGG2296. 3 Not detected Not detectedCTCGGGGGAGTTTGCACCaGG2297. 3 Not detected Not detectedGTGTTGGGAGTCTGCTCCaGG2298. 3 Not detected Not detectedGAGGGGGCAGGTTGCTCCaGG2299. 3 Not detected Not detectedGAGGGGAGAGTTTGTTCCaGG2300. 3 Not detected Not detectedGTGGCTGGAGTTTGCTGCtGG2301. 3 Not detected Not detectedGTCGGGGGAGTGGGCTCCaGG2302. 3 Not detected Not detected GAGGGGGGAGTGTGTTCCgGG 2303. 3 Not detected Not detected GTGGTGGGAGCTTGTTCCtGG 2304. 3 Not detected Not detectedGTGGGGGGTGCCTGCTCCaGG2305. 3 Not detected Not detected VEGFA Site 3 GAGTGAGTGTGTGCGTGtGG 2306.(on-target) 50.49 ± 1.25 20.05 ±0.01 GAGTGAGTGTGTGCGTGtGG 2307. 1 Not detected Not detected GTGTGAGTGTGTGCGTGgGG 2308. 1 Not detected Not detectedGTGTGAGTGTGTGCGTGaGG2309. 1 Not detected Not detectedGTGTGAGTGTGTGCGTGtGG2310. 1 Not detected Not detected GAGTGTGTGTGTGCGTGtGG 2311. 1 Not detected Not detected GAGTGGGTGTGTGCGTGgGG 2312. 1 Not detected Not detected GAGTGAGTGTGTGCGTGtGG 2313. 1 Not detected Not detected GAGTGAGTGTGTGGGTGgGG 2314. 1 Not detected Not detectedGAGTGAGTGTGTGTGTGtGG2315. 1 Not detected Not detected GAGTGAGTGTGTGTGTGtGG 2316. 1 Not detected Not detected PCT/US2014/029068 WO 2014/144592 118 Table 3CIndel mutation frequencies at potential off-target sites of tru-RGNs tarqeted to endogenous genes in human cellsTarget ID Target Site + PAMSEQ IDNumber of mismatchesIndel mutation frequency (%) ± s.e.m. GAGTGAGTGTGTGTGTGgGG 2317. 1 Not detected Not detected GAGTGAGTGTGTGTGTGtGG 2318. 1 Not detected Not detected GAGTGAGTGTGTGCGCGgGG 2319. 1 Not detected Not detected CTGTGAGTGTGTGCGTGaGG 2320. 2 Not detected Not detected ATGTGAGTGTGTGCGTGtGG 2321. 2 Not detected Not detected GCCTGAGTGTGTGCGTGtGG 2322. 2 Not detected Not detected GTGTGTGTGTGTGCGTGtGG 2323. 2 Not detected Not detected GTGTGGGTGTGTGCGTGtGG 2324. 2 Not detected Not detected GCGTGTGTGTGTGCGTGtGG 2325. 2 Not detected Not detected GTGTGTGTGTGTGCGTGgGG 2326. 2 Not detected Not detected GTGTGGGTGTGTGCGTGtGG 2327. 2 Not detected Not detectedGTGTGTGTGTGTGCGTGgGG 2328. 2 Not detected Not detectedGAGAGAGAGTGTGCGTGtGG2329. 2 Not detected Not detectedGAGTGTGTGAGTGCGTGgGG2330. 2 Not detected Not detectedGTGTGAGTGTGTGTGTGtGG2331. 2 Not detected Not detectedGAGTGTGTGTATGCGTGtGG 2332. 2 Not detected Not detectedGAGTCAGTGTGTGAGTGaGG2333. 2 Not detected Not detectedGAGTGTGTGTGTGAGTGtGG 2334. 2 Not detected Not detectedGAGTGTGTGTGTGCATGtGG2335. 2 Not detected Not detectedGAGTGAGAGTGTGTGTGtGG2336. 2 Not detected Not detectedGAGTGAGTGAGTGAGTGaGG 2337. 2 Not detected Not detected£714X7 site GTCCGAGCAGAAGAAGAAgGG 2338. 0 (on-target) 43.01 ± 0.87 17.25 + 0.64GTCTGAGCAGAAGAAGAAtGG2339. 1 Not detected Not detected GTCCCAGCAGTAGAAGAAtGG 2340. 2 Not detected Not detectedGTCCGAGGAGAGGAAGAAaGG2341. 2 Not detected Not detectedGTCAGAGGAGAAGAAGAAgGG2342. 2 Not detected Not detected GACAGAGCAGAAGAAGAAgGG 2343. 2 Not detected Not detectedGTGGGAGCAGAAGAAGAAgGG2344. 2 Not detected Not detected GTACTAGCAGAAGAAGAAaGG 2345. 2 Not detected Not detected GTCTGAGCAGAAGAAGAAtGG 2346. 2 Not detected Not detected GTGCTAGCAGAAGAAGAAgGG 2347. 2 Not detected Not detected TACAGAGCAGAAGAAGAAtGG 2348. 3 Not detected Not detected TACGGAGCAGAAGAAGAAtGG 2349. 3 Not detected Not detected AACGGAGCAGAAGAAGAAaGG 2350. 3 Not detected Not detected GACAGAGCAGAAGAAGAAgGG 2351. 3 Not detected Not detected PCT/US2014/029068 WO 2014/144592 119 Table 3CIndel mutation frequencies at potential off-target sites of tru-RGNs targeted to endogenous genes in human cellsTarget ID Target Site + PAMSEQ IDNumber of mismatchesIndel mutation frequency (%) ± s.e,m. CTGCGATCAGAAGAAGAAaGG 2352. 3 Not detected Not detected GACTGGGCAGAAGAAGAAgGG 2353. 3 Not detected Not detectedTTCCCTGCAGAAGAAGAAaGG2354. 3 Not detected Not detectedTTCCTACCAGAAGAAGAAtGG2355. 3 Not detected Not detected CTCTGAGGAGAAGAAGAAaGG 2356. 3 Not detected Not detected ATCCAATCAGAAGAAGAAgGG 2357. 3 Not detected Not detected GCCCCTGCAGAAGAAGAAcGG 2358. 3 Not detected Not detected ATCCAACCAGAAGAAGAAaGG 2359. 3 Not detected Not detected GACTGAGAAGAAGAAGAAaGG 2360. 3 Not detected Not detected CTGCGATCAGAAGAAGAAaGG 2361. 3 Not detected Not detected GACAGAGAAGAAGAAGAAaGG 2362. 3 Not detected Not detectedGTCATGGCAGAAGAAGAAaGG2363. 3 Not detected Not detectedGTTGGAGAAGAAGAAGAAgGG2364. 3 Not detected Not detected GTAAGAGAAGAAGAAGAAgGG 2365. 3 Not detected Not detected CTCCTAGCAAAAGAAGAAtGG 2366. 3 Not detected Not detected TTCAGAGCAGGAGAAGAAtGG 2367. 3 Not detected Not detected GTTGGAGCAGGAGAAGAAgGG 2368. 3 Not detected Not detected GCCTGAGCAGAAGGAGAAgGG 2369. 3 Not detected Not detected GTCTGAGGACAAGAAGAAtGG 2370. 3 Not detected Not detectedGTCCGGGAAGGAGAAGAAaGG2371. 3 Not detected Not detected GGCCGAGCAGAAGAAAGAcGG 2372. 3 Not detected Not detectedGTCCTAGCAGGAGAAGAAgAG2373. 3 Not detected Not detected WO 2014/144592 PCT/US2014/029068 120 Table 3D: Frequencies of tru-RGN-induced indel mutations at potential off- target sites in human U2OS.EGFP as determined by deep sequencing On- target siteOff-target site sequenceS#tru-RGN ControlInd eiWT Freq. Indel WT Freq2374. 150225640 0.66% 3 135451 0.00%GTGGGGGGAGTTTGCCCCaGG 0 GTGGGGGGTGTTTGCTCCCGG2375. 155152386 1.01% 0 86206 0.00%GTGGGTGGAGTTTGCTACtGG2376.471818 0.00% 0 199581 0.00% VEGFA GTGGGTGGAGTTTGCTACaGG2377.337298 0.00% 1 211547 0.00%site 1■2378.GTGGGTGGCGTTTGCTCCaGG210174 0.00% 1 105531 0.00%GTGTGGGGAATTTGCTCCaGG2379.673 715547 0.09% 1 387097 0.00%GTGGGGGGAGCTTTCTCCtGG2380.107757 0.00% 1 58735 0.00% GGGGGGGCAGTTTGCTCCtGG2381. 191566548 0.34% 3 297083 0.00% GTGTGAGTGTGTGCGTGtGG2382.324881 0.02% 9 122216 0.01%GTGTGAGTGTGTGCGTGaGG2383.532 194914 0.27% 11 73644 0.01%GAGTGGGTGTGTGCGTGgGG2384.237029 0.03% 10 178258 0.01%GAGTGACTGTGTGCGTGtGG2385.391894 0.00% 0 239460 0.00%GAGTGAGTGTGTGGGTGgGG2386.160140 0.01% 10 123324 0.01%GTGTGAGTGTGTGCGTGgGG2387.138687 0.01% 1 196271 0.00% VEGFA CAGTGAGTGTGTGCGTGtGG2388.546865 0.01% 41 355953 0.01%site 3GTGTGAGTGTGTGCGTGtGG2389.128 377451 0.03% 56 133978 0.04%GAGTGTGTGTGTGCGTGtGG2390.913 263028 0.35% 78 178979 0.04%GAGTGAGTGTGTGTGTGtGG2391.106933 0.04% 36 58812 0.06%GAGTGAGTGTGTGTGTGtGG2392.681 762999 0.09% 63 222451 0.03%GAGTGAGTGTGTGTGTGgGG2393,331 220289 0.15% 100 113911 0.09%GAGTGAGTGTGTGTGTGtGG2394.35725 0.00% 8 186495 0.00%GAGTGAGTGTGTGCGCGgGG2395.246893 0.04% 16 107623 0.01% GTCAGAGGAGAAGAAGAAgGG2396.201483 0.00% 4 148416 0.00%GTCAGAGGAGAAGAAGAAgGG2397.545662 0.00% 5 390884 0.00%GTCTGAGCACAAGAAGAAtGG2398.274212 0.00% 0 193837 0,00% EMX1 GTCTGAGCAGAAGAAGAAtGG2399.440 375646 0.12% 10 256181 0.00%site 1GACAGAGCAGAAGAAGAAgGG2400.212472 0.00% 1 158860 0.00%GTACTAGCAGAAGAAGAAaGG2401.152 229209 0.07% 103 157717 0.07%GTGGGAGCAGAAGAAGAAgGG2402.207401 0.02% 36 111183 0.03%GTCCCAGCAGTAGAAGAAtGG2403.226477 0.00% 1 278948 0.00% S#: SEQ ID NO: PCT/US2014/029068 WO 2014/144592 121 Example 2d. tru-gRNAs can be used with dual Cas9 nickases to efficiently induce genome editing in human cells tru-gRNAs were tested with the recently described dual Cas9 nickase approach to induce indel mutations. To do this, the Cas9-D10A nickase together with two full-length gRNAs targeted to sites in the human VEGFA gene (VEGFA site 1 and an additional sequence we refer to as VEGFA site 4) were co-expressed in U2OS.EGFP cells (Figure 4A).As described previously (Ran et al., 2013), this pair of nickases functioned cooperatively to induce high rates of indel mutations at the VEGFA target locus (Figure 4B).Interestingly, Cas9-D10A nickase co-expressed with only the gRNA targeted to VEGFA site 4 also induced indel mutations at a high frequency, albeit at a rate somewhat lower than that observed with the paired full- length gRNAs (Figure 4B).Importantly, use of a tru-gRNA for VEGFA site 1 in place of a full-length gRNA did not affect the efficacy of the dual nickase approach to induce indel mutations (Figure 4B). The dual nickase strategy has also been used to stimulate the introduction of specific sequence changes using ssODNs (Mali et al., 2013a; Ran et al., 2013) and so whether tru-gRNAs might be used for this type of alteration was also tested. Paired full-length gRNAs for VEGFA sites 1 and 4 together with Cas9-D10A nickase cooperatively enhanced efficient introduction of a short insertion from a ssODN donor (Figure 3A)into the VEGFA locus in human U2OS.EGFP cells as expected (Figure 3C).Again, the efficiency of ssODN-mediated sequence alteration by dual nicking remained equally high with the use of a tru-gRNA in place of the full-length gRNA targeted to VEGFA site 1 (Figure 3C).Taken together, these results demonstrate that tru-gRNAs can be utilized as part of a dual Cas9 nickase strategy to induce both indel mutations and ssODN-mediated sequence changes, without compromising the efficiency of genome editing by this approach.Having established that use of a tru-gRNA does not diminish the on-target genome editing activities of paired nickases, we next used deep sequencing to examine mutation frequencies at four previously identified bona fide off-target sites of the VEGFA site 1 gRNA. This analysis revealed that mutation rates dropped to essentially undetectable levels at all four of these off-target sites when using paired nickases with a tru-gRNA (Table 4). Bycontrast, neither a tru-RGN (Table 3B)nor the paired nickases with full-length gRNAs (Table 4)was able to completely eliminate off-target mutations at one of these four off-target sites (OT1-3). These WO 2014/144592 PCT/US2014/029068 results demonstrate that the use of tru-gRNAs can further reduce the off-target effects of paired Cas9 nickases (and vice versa) without compromising the efficiency of on- target genome editing.

Table 4 Frequencies of paired nickase-induced indel mutations at on- and off-target sites of VEGFA site 1 using full-length and tru-gRNAs Site Paired full-length gRNAs tru-gRNA/full-length gRNA Control Indel WT Freq. Indel WT Freq. Indel WT Freq. VEGFA site 1 78905 345696 18.583% 65754 280720 18.978% 170 308478 0.055% OT1-3 184 85151 0.216% 0 78658 0.000% 2 107850 0.002% OT1-4 0 89209 0.000% 1 97010 0.001% 0 102135 0.000% OT1-6 2 226575 0.001% 0 208218 0.000% 0 254580 0.000% OT1-11 0 124729 0.000% 0 121581 0.000% 0 155173 0.000% 5 WO 2014/144592 123 PCT/US2014/029068 References Cheng, A.W., Wang, H., Yang, H., Shi, L., Katz, Y., Theunissen, T.W., Rangarajan, S., Shivalila, C.S., Dadon, D.B., and Jaenisch, R. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res 23, 1163-1171. (2013).Cho, S.W., Kim, S., Kirn, J.M. & Kim, J.S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31, 230-232 (2013). Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339,819-823 (2013).Cradick, T.J., Fine, E.J., Antico, C.J., and Bao, G. CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. (2013).Dicarlo, J.E. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR- Cas systems. Nucleic Acids Res (2013).Ding, Q., Regan, S.N., Xia, Y., Oostrom, L.A., Cowan, C.A., and Musunuru, K. Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs Cell Stem Cell 12, 393-394. (2013).Fisher, S., Barry, A., Abreu, J., Minie, B., Nolan, J., Delorey, T.M., Young, G., Fennell, T.J., Allen, A., Ambrogio, L., et al. A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries. Genome Biol 12, RI. (2011).Friedland, A.E., Tzur, Y.B., Esvelt, K.M., Colaiacovo, M.P., Church, G.M., and Calarco, J.A. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods 10, 741-743. (2013).Fu, Y., Foden, J.A., Khayter, C., Maeder, M.L., Reyon, D., Joung, J.K., and Sander, J.D, High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31, 822-826. (2013).Gabriel, R. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol 29, 816-823 (2011).Gilbert, L.A., Larson, M.H., Morsut, L., Liu, Z., Brar, G.A., Torres, S.E., Stem- Ginossar, N., Brandman, O., Whitehead, E.H., Doudna, J,A., et al. (2013). CRISPR- Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell 154, 442-451.

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Claims (18)

1.289396/

2.CLAIMSWhat is claimed is: 1. A Cas9 guide RNA molecule for use in a method of increasing specificity of RNA-guided genome editing in a cell, the method comprising contacting the cell with a Casguide RNA molecule having a target complementarity region consisting of 17-nucleotides that are complementary to 17 -18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, wherein the target sequence is immediately 5’ of a protospacer adjacent motif (PAM). 2. A Cas9 guide RNA molecule for use in a method of inducing a break in a target region of a double-stranded DNA molecule in a cell and increasing specificity of RNA-guided genome editing in a cell, the method comprising expressing in or introducing into the cell: a Cas9 nuclease or a Cas9 nickase; and a Cas9 guide RNA molecule having a target complementarity region consisting of 17-nucleotides that are complementary to 17 -18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, wherein the target sequence is immediately 5’ of a protospacer adjacent motif (PAM).

3. The Cas9 guide RNA molecule for use of claim 1 or claim 2, wherein the Cas9 gRNA consists of the sequence: (X17-18)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC, wherein X17-18 is the target complementarity region consisting of 17-18 nucleotides that are complementary to 17 -consecutive nucleotides of the complementary strand of the selected target genomic sequence.

4. The Cas9 guide RNA molecule for use of claim 1 or claim 2, wherein the Cas9 gRNA consists of the sequence: (X17-18) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC 128 289396/ UUGAAAAAGUGGCACCGAGUCGGUGCUUUU, wherein X17-18 is the target complementarity region consisting of 17-18 nucleotides that are complementary to 17 -consecutive nucleotides of the complementary strand of the selected target genomic sequence.

5. The Cas9 guide RNA molecule for use of any one of claims 1 to 4, wherein the guide RNA is (i) a single guide RNA that includes a complementarity region consisting of 17-nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, or (ii) a crRNA that includes a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA.

6. The Cas9 guide RNA molecule for use of any one of claims 1 to 5, wherein the ribonucleic acid includes one or more U at the 3’ end of the molecule.

7. The Cas9 guide RNA molecule for use of any one of claims 1 to 6, wherein the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a selected target sequence; or wherein the complementarity region is complementary to 18 consecutive nucleotides of the complementary strand of a selected target sequence.

8. The Cas9 guide RNA molecule for use of claim 1 or claim 2, wherein the guide RNA consists of the sequence: (X17 18)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG; (X17-18)GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUC; or (X17-18)GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAG UUAAAAUAAGGCUAGUCCGUUAUC, wherein X17-18 is the target complementarity region consisting of 17-18 nucleotides that are complementary to 17 -18 consecutive nucleotides of the complementary strand of the selected target genomic sequence. 129 289396/

9. A Cas9 guide RNA (gRNA) molecule that includes a complementarity region at the 5′ end of the gRNA consisting of 17-18 nucleotides that are complementary to 17-consecutive nucleotides of the complementary strand of a target genomic sequence, wherein the target genomic sequence is immediately 5′ of a protospacer adjacent motif, and wherein the gRNA is a single gRNA or a CRISPR RNA (crRNA).

10. A complex comprising: a Cas9 nuclease and the gRNA molecule of claim 9.

11. The gRNA of claim 9, or the complex of claim 10, wherein the complementarity region of the gRNA consists of 17 nucleotides that are complementary to 17 consecutive nucleotides of the complementary strand of the target genomic sequence.

12. The gRNA of claim 9, or the complex of claim 10, wherein the complementarity region of the gRNA consists of 18 nucleotides that are complementary to 18 consecutive nucleotides of the complementary strand of the target genomic sequence.

13. The gRNA of any one of claims 9 and 11 to 12, or the complex of any one of claims 10 to 12, comprising a ribonucleic acid consisting of (X17-18) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO:1), (X17-18)GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC (SEQ ID NO:2), (X17-18)GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC (SEQ ID NO:3), (X17-18)GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:4), (X17-18)GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:5), (X17-18)GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:6), or (X17- 130 289396/ 18)GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:7), wherein (X17-18) is the complementarity region.

14. The gRNA of any one of claims 9 and 11 to 13, or the complex of any one of claims 10 to 13, comprising one or more uracil (U) at the 3′ end of the molecule.

15. A DNA molecule encoding the gRNA of any one of claims 9 and 11 to 14.

16. A vector comprising the DNA molecule of claim 15.

17. A host cell expressing the vector of claim 16.

18. The host cell of claim 17, wherein the cell is a eukaryotic cell. For the Applicants REINHOLD COHN AND PARTNERS By: